Modulation of Notch3 expression

ABSTRACT

Compounds, compositions and methods are provided for modulating the expression of Notch3. The compositions comprise oligonucleotides, targeted to nucleic acid encoding Notch3. Methods of using these compounds for modulation of Notch3 expression and for diagnosis and treatment of disease associated with expression of Notch3 are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods for modulating the expression of Notch3. In particular, this invention relates to compounds, particularly oligonucleotide compounds, which, in preferred embodiments, hybridize with nucleic acid molecules encoding Notch3. Such compounds are shown herein to modulate the expression of Notch3.

BACKGROUND OF THE INVENTION

[0002] Intrinsic, cell-autonomous factors as well as non-autonomous, short-range and long-range signals guide cells through distinct developmental paths. An organism frequently uses the same signaling pathway within different cellular contexts to achieve unique developmental goals.

[0003] Notch signaling is an evolutionarily conserved mechanism used to control cell fates through local cell interactions. The gene encoding the original Notch receptor was discovered in Drosophila melanogaster due to the fact that partial loss of function of the gene results in notches at the wing margin (Artavanis-Tsakonas et al., Science, 1999, 284, 770-776). Signals transmitted through the Notch receptor, in combination with other cellular factors, influence differentiation, proliferation and apoptotic events at all stages of development (Artavanis-Tsakonas et al., Science, 1999, 284, 770-776).

[0004] Mature Notch proteins are heterodimeric receptors derived from the cleavage of Notch pre-proteins into an extracellular subunit containing multiple EGF-like repeats and a transmembrane subunit including the intracellular region (Blaumueller et al., Cell, 1997, 90, 281-291). Notch activation results from the binding of ligands expressed by neighboring cells or soluble ligands and signaling from activated Notch involves networks of transcription regulators (Artavanis-Tsakonas et al., Science, 1995, 268, 225-232).

[0005] In context of experimental cancer immunotherapy, the Notch signaling network is acquiring increasing importance for its possible roles in neoplastic cells and the immune system (Jang et al., Curr. Opin. Mol. Ther., 2000, 2, 55-65). Larsson et al. predicted that the human Notch genes are proto-oncogenes and candidates for sites of chromosome breakage in neoplasia-associated translocations (Larsson et al., Genomics, 1994, 24, 253-258).

[0006] Four mammalian Notch homologs have been identified and are designated Notch1, Notch2, Notch3 and Notch4. Human Notch3 (also known as Notch (Drosophila) homolog 3 and CADASIL) was identified and mapped to chromosome 19p13.2-p13.1, a region associated with neoplasia-associated translocations (Larsson et al., Genomics, 1994, 24, 253-258).

[0007] Disclosed and claimed in U.S. Pat. No. 5,789,195 are nucleic acid sequences encoding Notch genes. Antibodies to human Notch proteins are additionally provided (Artavanis-Tsakonas et al., 1998). Amino acid sequences of Notch genes and antibodies against Notch proteins are also disclosed and claimed in U.S. Pat. No. 6,090,922 (Artavanis-Tsakonas et al., 2000).

[0008] Mutations in Notch3 have been identified as the cause of CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy), an autosomal dominant condition whose key features include recurrent subcortical ischemic strokes which lead to progressive dementia (Joutel and Tournier-Lasserve, Semin. Cell Dev. Biol., 1998, 9, 619-625).

[0009] Modulation of expression of Notch genes may prove to be a useful point for therapeutic intervention in developmental, hyperproliferative or autoimmune disorders or disorders arising from aberrant apoptosis.

[0010] Methods for genotypic diagnosis of CADASIL wherein the DNA of a symptomatic or at risk individual is contacted with primer pairs which specifically hybridize to Notch3 (Joutel et al., 1997).

[0011] Therapeutic methods utilizing anti-Notch3 antibodies and antisense sequences which block expression of Notch3 are disclosed and claimed in PCT publication WO 98/05775 (Tournier-Lasserve et al., 1998).

[0012] Methods for producing allergen- or antigen-tolerant T-cells employing compositions capable of upregulating expression of an endogenous Notch protein are disclosed and claimed in PCT publication WO 00/36089 (Lamb et al., 2000).

[0013] Disclosed and claimed in U.S. Pat. No. 6,149,902 is a method for cell transplantation which includes contacting a precursor cell with an agonist of Notch function effective to inhibit differentiation of the cell wherein said agonist is a Delta protein, a Serrate protein or an antibody to a Notch protein (Artavanis-Tsakonas et al., 2000).

[0014] Disclosed in U.S. Pat. No. 6,083,904 and PCT publication WO 94/07474 are therapeutic and diagnostic methods and compositions based on Notch proteins and nucleic acids, wherein antisense methods are generally disclosed (Artavanis-Tsakonas, 2000; Artavanis-Tsakonas et al., 1994).

[0015] Disclosed and claimed in U.S. Pat. No. 5,786,158 are methods and compositions for the detection of malignancy or nervous system disorders based on the level of Notch proteins or nucleic acids (Artavanis-Tsakonas et al., 1998).

[0016] Disclosed and claimed in PCT publication WO 00/20576 are methods for inducing differentiation and apoptosis in human cells that over express Notch proteins wherein Notch function is disrupted using antisense oligonucleotides that target the EGF repeat region, the lin/notch region and the ankyrin region (Miele et al., 2000).

[0017] Disclosed and claimed in PCT publication WO 01/25422 is an antisense oligonucleotide directed to exon 30 of human Notch3 (Bartelmez and Iversen, 2001).

[0018] Currently, there are no known therapeutic agents that effectively inhibit the synthesis of Notch3. To date, investigative strategies aimed at modulating Notch3 expression have involved the use of antibodies and Notch-regulating proteins as well as antisense RNA and oligonucleotides. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting Notch3 function.

[0019] Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of expression of Notch3.

[0020] The present invention provides compositions and methods for modulating expression of Notch3.

SUMMARY OF THE INVENTION

[0021] The present invention is directed to compounds, especially nucleic acid and nucleic acid-like oligomers, which are targeted to a nucleic acid encoding Notch3, and which modulate the expression of Notch3. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of screening for modulators of Notch3 and methods of modulating the expression of Notch3 in cells, tissues or animals comprising contacting said cells, tissues or animals with one or more of the compounds or compositions of the invention. Methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of Notch3 are also set forth herein. Such methods comprise administering a therapeutically or prophylactically effective amount of one or more of the compounds or compositions of the invention to the person in need of treatment.

DETAILED DESCRIPTION OF THE INVENTION

[0022] A. Overview of the Invention

[0023] The present invention employs compounds, preferably oligonucleotides and similar species for use in modulating the function or effect of nucleic acid molecules encoding Notch3. This is accomplished by providing oligonucleotides which specifically hybridize with one or more nucleic acid molecules encoding Notch3. As used herein, the terms “target nucleic acid” and “nucleic acid molecule encoding Notch3” have been used for convenience to encompass DNA encoding Notch3, RNA (including pre-mRNA and mRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA. The hybridization of a compound of this invention with its target nucleic acid is generally referred to as “antisense”. Consequently, the preferred mechanism believed to be included in the practice of some preferred embodiments of the invention is referred to herein as “antisense inhibition.” Such antisense inhibition is typically based upon hydrogen bonding-based hybridization of oligonucleotide strands or segments such that at least one strand or segment is cleaved, degraded, or otherwise rendered inoperable. In this regard, it is presently preferred to target specific nucleic acid molecules and their functions for such antisense inhibition.

[0024] The functions of DNA to be interfered with can include replication and transcription. Replication and transcription, for example, can be from an endogenous cellular template, a vector, a plasmid construct or otherwise. The functions of RNA to be interfered with can include functions such as translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA. One preferred result of such interference with target nucleic acid function is modulation of the expression of Notch3. In the context of the present invention, “modulation” and “modulation of expression” mean either an increase (stimulation) or a decrease (inhibition) in the amount or levels of a nucleic acid molecule encoding the gene, e.g., DNA or RNA. Inhibition is often the preferred form of modulation of expression and mRNA is often a preferred target nucleic acid.

[0025] In the context of this invention, “hybridization” means the pairing of complementary strands of oligomeric compounds. In the present invention, the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.

[0026] An antisense compound is specifically hybridizable when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.

[0027] In the present invention the phrase “stringent hybridization conditions” or “stringent conditions” refers to conditions under which a compound of the invention will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances and in the context of this invention, “stringent conditions” under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in which they are being investigated.

[0028] “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleobases of an oligomeric compound. For example, if a nucleobase at a certain position of an oligonucleotide (an oligomeric compound), is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, said target nucleic acid being a DNA, RNA, or oligonucleotide molecule, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The oligonucleotide and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other. Thus, “specifically hybridizable” and complementary are terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleobases such that stable and specific binding occurs between the oligonucleotide and a target nucleic acid.

[0029] It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). It is preferred that the antisense compounds of the present invention comprise at least 70% sequence complementarity to a target region within the target nucleic acid, more preferably that they comprise 90% sequence complementarity and even more preferably comprise 95% sequence complementarity to the target region within the target nucleic acid sequence to which they are targeted. For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0030] B. Compounds of the Invention

[0031] According to the present invention, compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges or loops. Once introduced to a system, the compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect modification of the target nucleic acid. One non-limiting example of such an enzyme is RNAse H, a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are “DNA-like” elicit RNAse H. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases such as those in the RNase III and ribonuclease L family of enzymes.

[0032] While the preferred form of antisense compound is a single-stranded antisense oligonucleotide, in many species the introduction of double-stranded structures, such as double-stranded RNA (dsRNA) molecules, has been shown to induce potent and specific antisense-mediated reduction of the function of a gene or its associated gene products. This phenomenon occurs in both plants and animals and is believed to have an evolutionary connection to viral defense and transposon silencing.

[0033] The first evidence that dsRNA could lead to gene silencing in animals came in 1995 from work in the nematode, Caenorhabditis elegans (Guo and Kempheus, Cell, 1995, 81, 611-620). Montgomery et al. have shown that the primary interference effects of dsRNA are posttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507). The posttranscriptional antisense mechanism defined in Caenorhabditis elegans resulting from exposure to double-stranded RNA (dsRNA) has since been designated RNA interference (RNAi). This term has been generalized to mean antisense-mediated gene silencing involving the introduction of dsRNA leading to the sequence-specific reduction of endogenous targeted mRNA levels (Fire et al., Nature, 1998, 391, 806-811). Recently, it has been shown that it is, in fact, the single-stranded RNA oligomers of antisense polarity of the dsRNAs which are the potent inducers of RNAi (Tijsterman et al., Science, 2002, 295, 694-697).

[0034] In the context of this invention, the term “oligomeric compound” refers to a polymer or oligomer comprising a plurality of monomeric units. In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologs thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.

[0035] While oligonucleotides are a preferred form of the compounds of this invention, the present invention comprehends other families of compounds as well, including but not limited to oligonucleotide analogs and mimetics such as those described herein.

[0036] The compounds in accordance with this invention preferably comprise from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides). One of ordinary skill in the art will appreciate that the invention embodies compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases in length.

[0037] In one preferred embodiment, the compounds of the invention are 12 to 50 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases in length.

[0038] In another preferred embodiment, the compounds of the invention are 15 to 30 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length.

[0039] Particularly preferred compounds are oligonucleotides from about 12 to about 50 nucleobases, even more preferably those comprising from about 15 to about 30 nucleobases.

[0040] Antisense compounds 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well.

[0041] Exemplary preferred antisense compounds include oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately upstream of the 5′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 8 to about 80 nucleobases). Similarly preferred antisense compounds are represented by oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately downstream of the 3′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 8 to about 80 nucleobases). One having skill in the art armed with the preferred antisense compounds illustrated herein will be able, without undue experimentation, to identify further preferred antisense compounds.

[0042] C. Targets of the Invention

[0043] “Targeting” an antisense compound to a particular nucleic acid molecule, in the context of this invention, can be a multistep process. The process usually begins with the identification of a target nucleic acid whose function is to be modulated. This target nucleic acid may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target nucleic acid encodes Notch3.

[0044] The targeting process usually also includes determination of at least one target region, segment, or site within the target nucleic acid for the antisense interaction to occur such that the desired effect, e.g., modulation of expression, will result. Within the context of the present invention, the term “region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic. Within regions of target nucleic acids are segments. “Segments” are defined as smaller or sub-portions of regions within a target nucleic acid. “Sites,” as used in the present invention, are defined as positions within a target nucleic acid.

[0045] Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon”. A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA transcribed from a gene encoding Notch3, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).

[0046] The terms “start codon region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon. Consequently, the “start codon region” (or “translation initiation codon region”) and the “stop codon region” (or “translation termination codon region”) are all regions which may be targeted effectively with the antisense compounds of the present invention.

[0047] The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Within the context of the present invention, a preferred region is the intragenic region encompassing the translation initiation or termination codon of the open reading frame (ORF) of a gene.

[0048] Other target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA (or corresponding nucleotides on the gene), and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA (or corresponding nucleotides on the gene). The 5′ cap site of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap site. It is also preferred to target the 5′ cap region.

[0049] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. Targeting splice sites, i.e., intron-exon junctions or exon-intron junctions, may also be particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred target sites. MRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as “fusion transcripts”. It is also known that introns can be effectively targeted using antisense compounds targeted to, for example, DNA or pre-mRNA.

[0050] It is also known in the art that alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as “variants”. More specifically, “pre-mRNA variants” are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequence.

[0051] Upon excision of one or more exon or intron regions, or portions thereof during splicing, pre-mRNA variants produce smaller “mRNA variants”. Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as “alternative splice variants”. If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.

[0052] It is also known in the art that variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more that one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as “alternative start variants” of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as “alternative stop variants” of that pre-mRNA or mRNA. One specific type of alternative stop variant is the “polyA variant” in which the multiple transcripts produced result from the alternative selection of one of the “polyA stop signals” by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites. Within the context of the invention, the types of variants described herein are also preferred target nucleic acids.

[0053] The locations on the target nucleic acid to which the preferred antisense compounds hybridize are hereinbelow referred to as “preferred target segments.” As used herein the term “preferred target segment” is defined as at least an 8-nucleobase portion of a target region to which an active antisense compound is targeted. While not wishing to be bound by theory, it is presently believed that these target segments represent portions of the target nucleic acid which are accessible for hybridization.

[0054] While the specific sequences of certain preferred target segments are set forth herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional preferred target segments may be identified by one having ordinary skill.

[0055] Target segments 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative preferred target segments are considered to be suitable for targeting as well.

[0056] Target segments can include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5′-terminus of the target segment and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). Similarly preferred target segments are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3′-terminus of the target segment and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). One having skill in the art armed with the preferred target segments illustrated herein will be able, without undue experimentation, to identify further preferred target segments.

[0057] Once one or more target regions, segments or sites have been identified, antisense compounds are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.

[0058] D. Screening and Target Validation

[0059] In a further embodiment, the “preferred target segments” identified herein may be employed in a screen for additional compounds that modulate the expression of Notch3. “Modulators” are those compounds that decrease or increase the expression of a nucleic acid molecule encoding Notch3 and which comprise at least an 8-nucleobase portion which is complementary to a preferred target segment. The screening method comprises the steps of contacting a preferred target segment of a nucleic acid molecule encoding Notch3 with one or more candidate modulators, and selecting for one or more candidate modulators which decrease or increase the expression of a nucleic acid molecule encoding Notch3. Once it is shown that the candidate modulator or modulators are capable of modulating (e.g. either decreasing or increasing) the expression of a nucleic acid molecule encoding Notch3, the modulator may then be employed in further investigative studies of the function of Notch3, or for use as a research, diagnostic, or therapeutic agent in accordance with the present invention.

[0060] The preferred target segments of the present invention may be also be combined with their respective complementary antisense compounds of the present invention to form stabilized double-stranded (duplexed) oligonucleotides.

[0061] Such double stranded oligonucleotide moieties have been shown in the art to modulate target expression and regulate translation as well as RNA processsing via an antisense mechanism. Moreover, the double-stranded moieties may be subject to chemical modifications (Fire et al., Nature, 1998, 391, 806-811; Timmons and Fire, Nature 1998, 395, 854; Timmons et al., Gene, 2001, 263, 103-112; Tabara et al., Science, 1998, 282, 430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507; Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir et al., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15, 188-200). For example, such double-stranded moieties have been shown to inhibit the target by the classical hybridization of antisense strand of the duplex to the target, thereby triggering enzymatic degradation of the target (Tijsterman et al., Science, 2002, 295, 694-697).

[0062] The compounds of the present invention can also be applied in the areas of drug discovery and target validation. The present invention comprehends the use of the compounds and preferred target segments identified herein in drug discovery efforts to elucidate relationships that exist between Notch3 and a disease state, phenotype, or condition. These methods include detecting or modulating Notch3 comprising contacting a sample, tissue, cell, or organism with the compounds of the present invention, measuring the nucleic acid or protein level of Notch3 and/or a related phenotypic or chemical endpoint at some time after treatment, and optionally comparing the measured value to a non-treated sample or sample treated with a further compound of the invention. These methods can also be performed in parallel or in combination with other experiments to determine the function of unknown genes for the process of target validation or to determine the validity of a particular gene product as a target for treatment or prevention of a particular disease, condition, or phenotype.

[0063] E. Kits, Research Reagents, Diagnostics, and Therapeutics

[0064] The compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. Furthermore, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes or to distinguish between functions of various members of a biological pathway.

[0065] For use in kits and diagnostics, the compounds of the present invention, either alone or in combination with other compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.

[0066] As one nonlimiting example, expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns.

[0067] Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression) (Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometry methods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

[0068] The compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding Notch3. For example, oligonucleotides that are shown to hybridize with such efficiency and under such conditions as disclosed herein as to be effective Notch3 inhibitors will also be effective primers or probes under conditions favoring gene amplification or detection, respectively. These primers and probes are useful in methods requiring the specific detection of nucleic acid molecules encoding Notch3 and in the amplification of said nucleic acid molecules for detection or for use in further studies of Notch3. Hybridization of the antisense oligonucleotides, particularly the primers and probes, of the invention with a nucleic acid encoding Notch3 can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of Notch3 in a sample may also be prepared.

[0069] The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense compounds have been employed as therapeutic moieties in the treatment of disease states in animals, including humans. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that antisense compounds can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.

[0070] For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of Notch3 is treated by administering antisense compounds in accordance with this invention. For example, in one non-limiting embodiment, the methods comprise the step of administering to the animal in need of treatment, a therapeutically effective amount of a Notch3 inhibitor. The Notch3 inhibitors of the present invention effectively inhibit the activity of the Notch3 protein or inhibit the expression of the Notch3 protein. In one embodiment, the activity or expression of Notch3 in an animal is inhibited by about 10%. Preferably, the activity or expression of Notch3 in an animal is inhibited by about 30%. More preferably, the activity or expression of Notch3 in an animal is inhibited by 50% or more.

[0071] For example, the reduction of the expression of Notch3 may be measured in serum, adipose tissue, liver or any other body fluid, tissue or organ of the animal. Preferably, the cells contained within said fluids, tissues or organs being analyzed contain a nucleic acid molecule encoding Notch3 protein and/or the Notch3 protein itself.

[0072] The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the compounds and methods of the invention may also be useful prophylactically.

[0073] F. Modifications

[0074] As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound, however, linear compounds are generally preferred. In addition, linear compounds may have internal nucleobase complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

[0075] Modified Internucleoside Linkages (Backbones)

[0076] Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[0077] Preferred modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

[0078] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0079] Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

[0080] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0081] Modified Sugar and Internucleoside Linkages-Mimetics

[0082] In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage (i.e. the backbone), of the nucleotide units are replaced with novel groups. The nucleobase units are maintained for hybridization with an appropriate target nucleic acid. One such compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0083] Preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

[0084] Modified Sugars

[0085] Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

[0086] Other preferred modifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl (2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. No. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0087] A further preferred modification of the sugar includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. The linkage is preferably a methelyne (—CH₂—)_(n) group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.

[0088] Natural and Modified Nucleobases

[0089] Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C═C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. , ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

[0090] Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, which is commonly owned with the instant application and also herein incorporated by reference.

[0091] Conjugates

[0092] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, the entire disclosure of which are incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.

[0093] Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

[0094] Chimeric Compounds

[0095] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide.

[0096] The present invention also includes antisense compounds which are chimeric compounds. “Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, increased stability and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAse H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. The cleavage of RNA:RNA hybrids can, in like fashion, be accomplished through the actions of endoribonucleases, such as RNAseL which cleaves both cellular and viral RNA. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

[0097] Chimeric antisense compounds of the invention may be formed as composite structures of two or more or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

[0098] The term “prodrug” indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dece. 9, 1993 or in WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0099] The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. For oligonucleotides, preferred examples of pharmaceutically acceptable salts and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0100] The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

[0101] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0102] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0103] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations. The pharmaceutical compositions and formulations of the present invention may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients.

[0104] Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Microemulsions are included as an embodiment of the present invention. Emulsions and their uses are well known in the art and are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0105] Formulations of the present invention include liposomal formulations. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes which are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.

[0106] Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0107] The pharmaceutical formulations and compositions of the present invention may also include surfactants. The use of surfactants in drug products, formulations and in emulsions is well known in the art. Surfactants and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0108] In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs. Penetration enhancers may be classified as′ belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Penetration enhancers and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0109] One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.

[0110] Preferred formulations for topical administration include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA).

[0111] For topical or other administration, oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters, pharmaceutically acceptable salts thereof, and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999, which is incorporated herein by reference in its entirety.

[0112] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or nonaqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred bile acids/salts and fatty acids and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Oral formulations for oligonucleotides and their preparation are described in detail in U.S. applications Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/315,298 (filed May 20, 1999) and Ser. No. 10/071,822, filed Feb. 8, 2002, each of which is incorporated herein by reference in their entirety.

[0113] Compositions and formulations for parenteral, intra-thecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0114] Certain embodiments of the invention provide pharmaceutical compositions containing one or more oligomeric compounds and one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to cancer chemotherapeutic drugs such as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. Combinations of antisense compounds and other non-antisense drugs are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

[0115] In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Alternatively, compositions of the invention may contain two or more antisense compounds targeted to different regions of the same nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially.

[0116] H. Dosing

[0117] The formulation of therapeutic compositions and their subsequent administration (dosing) is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC₅₀s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.

[0118] While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same.

EXAMPLES Example 1 Synthesis of Nucleoside Phosphoramidites

[0119] The following compounds, including amidites and their intermediates were prepared as described in U.S. Pat. No. 6,426,220 and published PCT WO 02/36743; 5′-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for 5-methyl-dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine penultimate intermediate for 5-methyl dC amidite, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine, 2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modified amidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate, 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T amidite), 5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine intermediate, 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidine penultimate intermediate, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C amidite), [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE A amdite), [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and 2′-O-(dimethylaminooxyethyl) nucleoside amidites, 2′-(Dimethylaminooxyethoxy) nucleoside amidites, 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine, 5′-O-tert-Butyldiphenylsilyl-2′-O(2-hydroxyethyl)-5-methyluridine, 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine, 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine, 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N dimethylaminooxyethyl]-5-methyluridine, 2′-O-(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite], 2′-(Aminooxyethoxy) nucleoside amidites, N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite], 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites, 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine, 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine and 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 2 Oligonucleotide and Oligonucleoside Synthesis

[0120] The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.

[0121] Oligonucleotides: Unsubstituted and substituted phosphodiester (P═O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry with oxidation by iodine.

[0122] Phosphorothioates (P═S) are synthesized similar to phosphodiester oligonucleotides with the following exceptions: thiation was effected by utilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the oxidation of the phosphite linkages. The thiation reaction step time was increased to 180 sec and preceded by the normal capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (12-16 hr), the oligonucleotides were recovered by precipitating with >3 volumes of ethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.

[0123] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.

[0124] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporated by reference.

[0125] Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.

[0126] Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.

[0127] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.

[0128] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.

[0129] Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

[0130] Oligonucleosides: Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.

[0131] Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.

[0132] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 3 RNA Synthesis

[0133] In general, RNA synthesis chemistry is based on the selective incorporation of various protecting groups at strategic intermediary reactions. Although one of ordinary skill in the art will understand the use of protecting groups in organic synthesis, a useful class of protecting groups includes silyl ethers. In particular bulky silyl ethers are used to protect the 5′-hydroxyl in combination with an acid-labile orthoester protecting group on the 2′-hydroxyl. This set of protecting groups is then used with standard solid-phase synthesis technology. It is important to lastly remove the acid labile orthoester protecting group after all other synthetic steps. Moreover, the early use of the silyl protecting groups during synthesis ensures facile removal when desired, without undesired deprotection of 2′ hydroxyl.

[0134] Following this procedure for the sequential protection of the 5′-hydroxyl in combination with protection of the 2′-hydroxyl by protecting groups that are differentially removed and are differentially chemically labile, RNA oligonucleotides were synthesized.

[0135] RNA oligonucleotides are synthesized in a stepwise fashion. Each nucleotide is added sequentially (3′- to 5′-direction) to a solid support-bound oligonucleotide. The first nucleoside at the 3′-end of the chain is covalently attached to a solid support. The nucleotide precursor, a ribonucleoside phosphoramidite, and activator are added, coupling the second base onto the 5′-end of the first nucleoside. The support is washed and any unreacted 5′-hydroxyl groups are capped with acetic anhydride to yield 5′-acetyl moieties. The linkage is then oxidized to the more stable and ultimately desired P(V) linkage. At the end of the nucleotide addition cycle, the 5′-silyl group is cleaved with fluoride. The cycle is repeated for each subsequent nucleotide.

[0136] Following synthesis, the methyl protecting groups on the phosphates are cleaved in 30 minutes utilizing 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂) in DMF. The deprotection solution is washed from the solid support-bound oligonucleotide using water. The support is then treated with 40% methylamine in water for 10 minutes at 55° C. This releases the RNA oligonucleotides into solution, deprotects the exocyclic amines, and modifies the 2′-groups. The oligonucleotides can be analyzed by anion exchange HPLC at this stage.

[0137] The 2′-orthoester groups are the last protecting groups to be removed. The ethylene glycol monoacetate orthoester protecting group developed by Dharmacon Research, Inc. (Lafayette, Colo.), is one example of a useful orthoester protecting group which, has the following important properties. It is stable to the conditions of nucleoside phosphoramidite synthesis and oligonucleotide synthesis. However, after oligonucleotide synthesis the oligonucleotide is treated with methylamine which not only cleaves the oligonucleotide from the solid support but also removes the acetyl groups from the orthoesters. The resulting 2-ethyl-hydroxyl substituents on the orthoester are less electron withdrawing than the acetylated precursor. As a result, the modified orthoester becomes more labile to acid-catalyzed hydrolysis. Specifically, the rate of cleavage is approximately 10 times faster after the acetyl groups are removed. Therefore, this orthoester possesses sufficient stability in order to be compatible with oligonucleotide synthesis and yet, when subsequently modified, permits deprotection to be carried out under relatively mild aqueous conditions compatible with the final RNA oligonucleotide product.

[0138] Additionally, methods of RNA synthesis are well known in the art (Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe, S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M. D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191; Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22, 1859-1862; Dahl, B. J., et al., Acta Chem. Scand,. 1990, 44, 639-641; Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott, F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2315-2331).

[0139] RNA antisense compounds (RNA oligonucleotides) of the present invention can be synthesized by the methods herein or purchased from Dharmacon Research, Inc (Lafayette, Colo.). Once synthesized, complementary RNA antisense compounds can then be annealed by methods known in the art to form double stranded (duplexed) antisense compounds. For example, duplexes can be formed by combining 30 μl of each of the complementary strands of RNA oligonucleotides (50 um RNA oligonucleotide solution) and 15 μl of 5× annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90° C., then 1 hour at 37° C. The resulting duplexed antisense compounds can be used in kits, assays, screens, or other methods to investigate the role of a target nucleic acid.

Example 4 Synthesis of Chimeric Oligonucleotides

[0140] Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the “gap” segment of linked nucleosides is positioned between 5′ and 3′ “wing” segments of linked nucleosides and a second “open end” type wherein the “gap” segment is located at either the 3′ or the 5′ terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]--[2′-deoxy]--[2′-O-Me] Chimeric Phosphorothioate Oligonucleotides

[0141] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 394, as above. Oligonucleotides are synthesized using the automated synthesizer and 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphor-amidite for the DNA portion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings. The standard synthesis cycle is modified by incorporating coupling steps with increased reaction times for the 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protected oligonucleotide is cleaved from the support and deprotected in concentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotected oligo is then recovered by an appropriate method (precipitation, column chromatography, volume reduced in vacuo and analyzed spetrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]--[2′-deoxy]--[2′-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides

[0142] [2′-O-(2-methoxyethyl)]--[2′-deoxy]--[-2′-O-(methoxyethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2′-O-methyl chimeric oligonucleotide, with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]--[2′-deoxy Phosphorothioate]--[2′-O-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides

[0143] [2′-O-(2-methoxyethyl phosphodiester]--[2′-deoxy phosphorothioate]--[2′-O-(methoxyethyl) phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2′-O-methyl chimeric oligonucleotide with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidation with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.

[0144] Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5 Design and Screening of Duplexed Antisense Compounds Targeting Notch3

[0145] In accordance with the present invention, a series of nucleic acid duplexes comprising the antisense compounds of the present invention and their complements can be designed to target Notch3. The nucleobase sequence of the antisense strand of the duplex comprises at least a portion of an oligonucleotide in Table 1. The ends of the strands may be modified by the addition of one or more natural or modified nucleobases to form an overhang. The sense strand of the dsRNA is then designed and synthesized as the complement of the antisense strand and may also contain modifications or additions to either terminus. For example, in one embodiment, both strands of the dsRNA duplex would be complementary over the central nucleobases, each having overhangs at one or both termini.

[0146] For example, a duplex comprising an antisense strand having the sequence CGAGAGGCGGACGGGACCG and having a two-nucleobase overhang of deoxythymidine(dT) would have the following structure:   cgagaggcggacgggaccgTT Antisense Strand   ||||||||||||||||||| TTgctctccgcctgccctggc Complement

[0147] RNA strands of the duplex can be synthesized by methods disclosed herein or purchased from Dharmacon Research Inc., (Lafayette, Colo.). Once synthesized, the complementary strands are annealed. The single strands are aliquoted and diluted to a concentration of 50 uM. Once diluted, 30 uL of each strand is combined with 15uL of a 5× solution of annealing buffer. The final concentration of said buffer is 100 mm potassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The final volume is 75 uL. This solution is incubated for 1 minute at 90° C. and then centrifuged for 15 seconds. The tube is allowed to sit for 1 hour at 37° C. at which time the dsRNA duplexes are used in experimentation. The final concentration of the dsRNA duplex is 20 uM. This solution can be stored frozen (−20° C.) and freeze-thawed up to 5 times.

[0148] Once prepared, the duplexed antisense compounds are evaluated for their ability to modulate Notch3 expression.

[0149] When cells reached 80% confluency, they are treated with duplexed antisense compounds of the invention. For cells grown in 96-well plates, wells are washed once with 200 μL OPTI-MEM-1 reduced-serum medium (Gibco BRL) and then treated with 130 μL of OPTI-MEM-1 containing 12 μg/mL LIPOFECTIN (Gibco BRL) and the desired duplex antisense compound at a final concentration of 200 nm. After 5 hours of treatment, the medium is replaced with fresh medium. Cells are harvested 16 hours after treatment, at which time RNA is isolated and target reduction measured by RT-PCR.

Example 6 Oligonucleotide Isolation

[0150] After cleavage from the controlled pore glass solid support and deblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours, the oligonucleotides or oligonucleosides are recovered by precipitation out of 1 M NH₄OAc with >3 volumes of ethanol. Synthesized oligonucleotides were analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis and judged to be at least 70% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis was determined by the ratio of correct molecular weight relative to the −16 amu product (+/−32+/−48). For some studies oligonucleotides were purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.

Example 7 Oligonucleotide Synthesis—96 Well Plate Format

[0151] Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per standard or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.

[0152] Oligonucleotides were cleaved from support and deprotected with concentrated NH₄0H at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.

Example 8 Oligonucleotide Analysis—96-Well Plate Format

[0153] The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length.

Example 9 Cell Culture and Oligonucleotide Treatment

[0154] The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen. This can be readily determined by methods routine in the art, for example Northern blot analysis, ribonuclease protection assays, or RT-PCR.

[0155] T-24 Cells:

[0156] The human transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy's 5A basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #353872) at a density of 7000 cells/well for use in RT-PCR analysis.

[0157] For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

[0158] A549 Cells:

[0159] The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells were routinely cultured in DMEM basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.

[0160] NHDF Cells:

[0161] Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Corporation (Walkersville, MD). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier.

[0162] HEK Cells:

[0163] Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville, Md.). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier.

[0164] Treatment with Antisense Compounds:

[0165] When cells reached 65-75% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 100 μL OPTI-MEM™-1 reduced-serum medium (Invitrogen Corporation, Carlsbad, Calif.) and then treated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation, Carlsbad, Calif.) and the desired concentration of oligonucleotide. Cells are treated and data are obtained in triplicate. After 4-7 hours of treatment at 37° C., the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment.

[0166] The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide is selected from either ISIS 13920 (TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted to human Jun-N-terminal kinase-2 (JNK2). Both controls are 2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone. For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) or c-raf (for ISIS 15770) mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments. The concentrations of antisense oligonucleotides used herein are from 50 nM to 300 nM.

Example 10 Analysis of Oligonucleotide Inhibition of Notch3 Expression

[0167] Antisense modulation of Notch3 expression can be assayed in a variety of ways known in the art. For example, Notch3 mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. The preferred method of RNA analysis of the present invention is the use of total cellular RNA as described in other examples herein. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.

[0168] Protein levels of Notch3 can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA) or fluorescence-activated cell sorting (FACS). Antibodies directed to Notch3 can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art.

Example 11 Design of Phenotypic Assays and In Vivo Studies for the Use of Notch3 Inhibitors

[0169] Phenotypic Assays

[0170] Once Notch3 inhibitors have been identified by the methods disclosed herein, the compounds are further investigated in one or more phenotypic assays, each having measurable endpoints predictive of efficacy in the treatment of a particular disease state or condition. Phenotypic assays, kits and reagents for their use are well known to those skilled in the art and are herein used to investigate the role and/or association of Notch3 in health and disease. Representative phenotypic assays, which can be purchased from any one of several commercial vendors, include those for determining cell viability, cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assays including enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.), cell regulation, signal transduction, inflammation, oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube formation assays, cytokine and hormone assays and metabolic assays (Chemicon International Inc., Temecula, Calif.; Amersham Biosciences, Piscataway, N.J.).

[0171] In one non-limiting example, cells determined to be appropriate for a particular phenotypic assay (i.e., MCF-7 cells selected for breast cancer studies; adipocytes for obesity studies) are treated with Notch3 inhibitors identified from the in vitro studies as well as control compounds at optimal concentrations which are determined by the methods described above. At the end of the treatment period, treated and untreated cells are analyzed by one or more methods specific for the assay to determine phenotypic outcomes and endpoints.

[0172] Phenotypic endpoints include changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides or metals. Measurements of cellular status which include pH, stage of the cell cycle, intake or excretion of biological indicators by the cell, are also endpoints of interest.

[0173] Analysis of the geneotype of the cell (measurement of the expression of one or more of the genes of the cell) after treatment is also used as an indicator of the efficacy or potency of the Notch3 inhibitors. Hallmark genes, or those genes suspected to be associated with a specific disease state, condition, or phenotype, are measured in both treated and untreated cells.

[0174] In Vivo Studies

[0175] The individual subjects of the in vivo studies described herein are warm-blooded vertebrate animals, which includes humans.

[0176] The clinical trial is subjected to rigorous controls to ensure that individuals are not unnecessarily put at risk and that they are fully informed about their role in the study. To account for the psychological effects of receiving treatments, volunteers are randomly given placebo or Notch3 inhibitor. Furthermore, to prevent the doctors from being biased in treatments, they are not informed as to whether the medication they are administering is a Notch3 inhibitor or a placebo. Using this randomization approach, each volunteer has the same chance of being given either the new treatment or the placebo.

[0177] Volunteers receive either the Notch3 inhibitor or placebo for eight week period with biological parameters associated with the indicated disease state or condition being measured at the beginning (baseline measurements before any treatment), end (after the final treatment), and at regular intervals during the study period. Such measurements include the levels of nucleic acid molecules encoding Notch3 or Notch3 protein levels in body fluids, tissues or organs compared to pre-treatment levels. Other measurements include, but are not limited to, indices of the disease state or condition being treated, body weight, blood pressure, serum titers of pharmacologic indicators of disease or toxicity as well as ADME (absorption, distribution, metabolism and excretion) measurements.

[0178] Information recorded for each patient includes age (years), gender, height (cm), family history of disease state or condition (yes/no), motivation rating (some/moderate/great) and number and type of previous treatment regimens for the indicated disease or condition.

[0179] Volunteers taking part in this study are healthy adults (age 18 to 65 years) and roughly an equal number of males and females participate in the study. Volunteers with certain characteristics are equally distributed for placebo and Notch3 inhibitor treatment. In general, the volunteers treated with placebo have little or no response to treatment, whereas the volunteers treated with the Notch3 inhibitor show positive trends in their disease state or condition index at the conclusion of the study.

Example 12 RNA Isolation

[0180] Poly(A)+ mRNA Isolation

[0181] Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation are routine in the art. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C., was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.

[0182] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

[0183] Total RNA Isolation

[0184] Total RNA was isolated using an RNEASY ₉₆™ kit and buffers purchased from Qiagen Inc. (Valencia, Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 150 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 150 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY ₉₆™ well plate attached to a QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY ₉₆™ plate and incubated for 15 minutes and the vacuum was again applied for 1 minute. An additional 500 μL of Buffer RW1 was added to each well of the RNEASY ₉₆™ plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE was then added to each well of the RNEASY ₉₆™ plate and the vacuum applied for a period of 90 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 3 minutes. The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 140 μL of RNAse free water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.

[0185] The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.

Example 13 Real-Time Quantitative PCR Analysis of Notch3 mRNA Levels

[0186] Quantitation of Notch3 mRNA levels was accomplished by real-time quantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM™ Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.

[0187] Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only (“single-plexing”), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.

[0188] PCR reagents were obtained from Invitrogen Corporation, (Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail (2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of DATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-well plates containing 30 μL total RNA solution (20-200 ng). The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

[0189] Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen™ (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen™ RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA quantification by RiboGreen™ are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).

[0190] In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™ reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nm and emission at 530 nm.

[0191] Probes and primers to human Notch3 were designed to hybridize to a human Notch3 sequence, using published sequence information (the complement of residues 118831-163178 of GenBank accession number NT_(—)011290.3, representing a genomic sequence of Notch3, incorporated herein as SEQ ID NO: 4). For human Notch3 the PCR primers were: forward primer: TCACCATGCCGTAACGGG (SEQ ID NO: 5) reverse primer: TCGGTGTCCTGGACAGTCG (SEQ ID NO: 6) and the PCR probe was: FAM-CTTCCTGGGTTTGAGGGTCAGAATTGTG-TAMRA (SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCR primers were: forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:8) reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:9) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 10) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

Example 14 Northern Blot Analysis of Notch3 mRNA Levels

[0192] Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBOND™-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.

[0193] To detect human Notch3, a human Notch3 specific probe was prepared by PCR using the forward primer TCACCATGCCGTAACGGG (SEQ ID NO: 5) and the reverse primer TCGGTGTCCTGGACAGTCG (SEQ ID NO: 6). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0194] Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.

Example 15 Antisense Inhibition of Human Notch3 Expression by Chimeric Phosphorothioate Oigonucleotides Having 2′-MOE Wings and a Deoxy Gap

[0195] In accordance with the present invention, a series of antisense compounds were designed to target different regions of the human Notch3 RNA, using published sequences (the complement of residues 118831-163178 of GenBank accession number NT_(—)011290.3, representing a genomic sequence of Notch3, incorporated herein as SEQ ID NO: 4; GenBank accession number NM_(—)000435.1, incorporated herein as SEQ ID NO: 11). The compounds are shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the compound binds. All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on human Notch3 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from three experiments in which A549 cells were treated with the oligonucleotides of the present invention. The positive control for each datapoint is identified in the table by sequence ID number. If present, “N.D.” indicates “no data”. TABLE 1 Inhibition of human Notch3 mRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap TARGET CONTROL SEQ ID TARGET SEQ ID SEQ ID ISIS # REGION NO SITE SEQUENCE % INHIB NO NO 226740 Coding 4 1661 cccgctagcagcagcagcag 69 12 1 226741 Coding 4 4930 caacgacctccatttgcaca 59 13 1 226742 Coding 4 4935 gggtgcaacgacctccattt 17 14 1 226743 Coding 4 10042 gccaccactgaactctggca 77 15 1 226744 Coding 4 10638 ggacagtcgtccacgttcac 83 16 1 226745 Coding 4 10708 aggagggcactggcagttat 55 17 1 226746 Coding 4 10947 atattctgactgcagctctc 79 18 1 226747 Coding 11 1105 cacaggaggccagtcttgcc 51 19 1 226748 Coding 4 13104 tttgtgtcacagatagcatc 70 20 1 226749 Coding 4 13385 gtctcacagcgaggtccagt 92 21 1 226750 Coding 11 1450 gttcctgtgaagcctgccat 86 22 1 226751 Coding 4 14216 tgcagctgaagccattgact 78 23 1 226752 Coding 4 15268 ccacctggctctcgcagcgt 82 24 1 226753 Coding 4 15336 gcagaggtacttgtccacca 78 25 1 226754 Coding 4 15341 cagcggcagaggtacttgtc 56 26 1 226755 Coding 11 1912 tcgcagttcacacctgtggt 77 27 1 226756 Coding 4 15553 cggttgatgccatcacggca 68 28 1 226757 Coding 4 16814 cactcattgatctccacgtt 59 29 1 226758 Coding 4 16862 ttttccccatccacacagga 34 30 1 226759 Coding 4 18037 tccacatcctgctggcatcg 46 31 1 226760 Coding 4 20876 gtcgggcaggtcctgttcgc 65 32 1 226761 Coding 4 21386 ggcagtgggctcctgtgtag 66 33 1 226762 Coding 4 22235 cgcctgacacagctgctcca 82 34 1 226763 Coding 4 22284 ctgggcacacgcagtagtgg 55 35 1 226764 Coding 4 22372 atatagccacggcaggtccc 69 36 1 226765 Coding 11 3396 caggaagacactcacacatg 59 37 1 226766 Coding 4 23287 gaaaccacccaccaggtcca 77 38 1 226767 Coding 4 23385 cagtcccgggtgtgtgccgc 52 39 1 226768 Coding 4 23588 cggcactggcctccatgctg 98 40 1 226769 Coding 4 23627 caggtgaaggtcagcccacc 26 41 1 226770 Coding 4 23632 agtgacaggtgaaggtcagc 59 42 1 226771 Coding 4 24425 tgcagctcccggcaggagcg 51 43 1 226772 Coding 4 24430 ggcactgcagctcccggcag 65 44 1 226773 Coding 4 24764 cagcctgggctgttgcactc 69 45 1 226774 Coding 4 24861 gcagcggctgttgttgaaga 70 46 1 226775 Coding 4 28093 gtggtcggcgcagtacttct 54 47 1 226776 Coding 4 28098 gcaaagtggtcggcgcagta 77 48 1 226777 Coding 4 32045 aaccagagggtgctgtgctc 63 49 1 226778 Coding 11 5188 cccttggccatgttcttcat 83 50 1 226779 Intron: 4 32312 tctcacccttggccatgttc 86 51 1 Exon Junction 226780 Coding 4 32342 tccagtctgtggccacctcc 90 52 1 226781 Coding 11 5275 atgcctggctcctctacctt 78 53 1 226782 Coding 4 35155 agtgccatggctggtgccac 78 54 1 226783 Coding 4 36465 ctagctgatgtgtcatctgc 71 55 1 226784 Coding 4 36603 gtgtctgccccagcatccag 82 56 1 226785 Coding 4 37060 gcgatgagctcttccaccat 69 57 1 226786 Coding 4 37065 ggctggcgatgagctcttcc 28 58 1 226787 Coding 4 40803 gcttggcagcctcatagctg 36 59 1 226788 Coding 4 40808 cagcagcttggcagcctcat 80 60 1 226789 Coding 4 40920 cactgggttgatccagcaag 65 61 1 226790 Coding 4 41240 cactgcagtggcagtggcag 87 62 1 226791 Coding 4 41548 acccgcaggaagcgggcctt 47 63 1 226792 Coding 4 41553 tgggaacccgcaggaagcgg 6 64 1 226793 Coding 4 41637 cggaccagtctgagagggag 51 65 1 226794 Stop 4 41806 gcgtctcaggccaacacttg 76 66 1 Codon 226795 3′UTR 4 41826 caagatctaagaactgacga 75 67 1 226796 3′UTR 4 41936 caaggcaaggatgcaggagg 72 68 1 226797 3′UTR 4 42120 ttcatgtcagagtgtaagga 64 69 1 226798 3′UTR 4 42225 catatatatattttgtaaaa 21 70 1 226799 3′UTR 4 42280 cacagactcagccccacggg 67 71 1 226800 3′UTR 4 42302 atccagcttggccgaatggg 69 72 1 226801 3′UTR 4 42363 tacctgggtcgtgtactcgg 66 73 1 226802 3′UTR 4 42401 gccccagtgggtgcgcccaa 69 74 1 226803 3′UTR 4 42465 tggaatgcagtgaagtgagg 83 75 1 226804 3′UTR 4 42505 gtgggcccttccccagcaag 67 76 1 226805 3′UTR 4 42557 agttcccaaagggagatggc 71 77 1 226806 3′UTR 11 7852 tcccacatttacagggacat 63 78 1 226807 3′UTR 4 42651 tcaggcagctcctcttcttg 81 79 1 226808 3′UTR 4 42682 ccagaggattaccaggaaga 67 80 1 226809 3′UTR 4 42713 aaaaatacctctattctgcc 24 81 1 226810 Intron 4 3944 ggctggtggatcacctgagg 56 82 1 226811 Intron 4 15976 ctccgcctcctgaggtcaag 71 83 1 226812 Intron 4 20601 gggatatgccttggattgag 28 84 1 226813 Intron: 4 24948 atgggctcacttgcaagtgc 39 85 1 Exon Junction 226814 Intron: 4 28405 ggtcactcacccgatcacct 53 86 1 Exon Junction 226815 Intron: 4 35224 tgtcactaacctgggccacg 83 87 1 Exon Junction 226816 Intron: 4 36957 ggatgagaatctaggacaga 14 88 1 Exon Junction 226817 Intron 4 40290 ggttttactacgttggccag 78 89 1

[0196] As shown in Table 1, SEQ ID NOs 12, 13, 15, 16, 18, 20, 21, 22, 23, 24, 25, 27, 28, 29, 32, 33, 34, 36, 37, 38, 40, 42, 43, 44, 45, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 60, 61, 62, 66, 67, 68, 69, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 83, 87 and 89 demonstrated at least 59% inhibition of human Notch3 expression in this assay and are therefore preferred. More preferred are SEQ ID NOs: 21, 51 and 52. The target regions to which these preferred sequences are complementary are herein referred to as “preferred target segments” and are therefore preferred for targeting by compounds of the present invention. These preferred target segments are shown in Table 2. The sequences represent the reverse complement of the preferred antisense compounds shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target nucleic acid to which the oligonucleotide binds. Also shown in Table 2 is the species in which each of the preferred target segments was found. TABLE 2 Sequence and position of preferred target regions identified in Notch3. TARGET SEQ ID TARGET REV COMP SEQ ID SITEID NO SITE SEQUENCE OF SEQ ID ACTIVE IN NO 143392 4 1661 ctgctgctgctgctagcggg 12 H. sapiens 90 143393 4 4930 tgtgcaaatggaggtcgttg 13 H. sapiens 91 143395 4 10042 tgccagagttcagtggtggc 15 H. sapiens 92 143396 4 10638 gtgaacgtggacgactgtcc 16 H. sapiens 93 143398 4 10947 gagagctgcagtcagaatat 18 H. sapiens 94 143400 4 13104 gatgctatctgtgacacaaa 20 H. sapiens 95 143401 4 13385 actggacctcgctgtgagac 21 H. sapiens 96 143402 11 1450 atggcaggcttcacaggaac 22 H. sapiens 97 143403 4 14216 agtcaatggcttcagctgca 23 H. sapiens 98 143404 4 15268 acgctgcgagagccaggtgg 24 H. sapiens 99 143405 4 15336 tggtggacaagtacctctgc 25 H. sapiens 100 143407 11 1912 accacaggtgtgaactgcga 27 H. sapiens 101 143408 4 15553 tgccgtgatggcatcaaccg 28 H. sapiens 102 143409 4 16814 aacgtggagatcaatgagtg 29 H. sapiens 103 143412 4 20876 gcgaacaggacctgcccgac 32 H. sapiens 104 143413 4 21386 ctacacaggagcccactgcc 33 H. sapiens 105 143414 4 22235 tggagcagctgtgtcaggcg 34 H. sapiens 106 143416 4 22372 gggacctgccgtggctatat 36 H. sapiens 107 143417 11 3396 catgtgtgagtgtcttcctg 37 H. sapiens 108 143418 4 23287 tggacctggtgggtggtttc 38 H. sapiens 109 143420 4 23588 cagcatggaggccagtgccg 40 H. sapiens 110 143422 4 23632 gctgaccttcacctgtcact 42 H. sapiens 111 143424 4 24430 ctgccgggagctgcagtgcc 44 H. sapiens 112 143425 4 24764 gagtgcaacagcccaggctg 45 H. sapiens 113 143426 4 24861 tcttcaacaacagccgctgc 46 H. sapiens 114 143428 4 28098 tactgcgccgaccactttgc 48 H. sapiens 115 143429 4 32045 gagcacagcaccctctggtt 49 H. sapiens 116 143430 11 5188 atgaagaacatggccaaggg 50 H. sapiens 117 143431 4 32312 gaacatggccaagggtgaga 51 H. sapiens 118 143432 4 32342 ggaggtggccacagactgga 52 H. sapiens 119 143433 11 5275 aaggtagaggagccaggcat 53 H. sapiens 120 143434 4 35155 gtggcaccagccatggcact 54 H. sapiens 121 143435 4 36465 gcagatgacacatcagctag 55 H. sapiens 122 143436 4 36603 ctggatgctggggcagacac 56 H. sapiens 123 143437 4 37060 atggtggaagagctcatcgc 57 H. sapiens 124 143440 4 40808 atgaggctgccaagctgctg 60 H. sapiens 125 143441 4 40920 cttgctggatcaacccagtg 61 H. sapiens 126 143442 4 41240 ctgccactgccactgcagtg 62 H. sapiens 127 143446 4 41806 caagtgttggcctgagacgc 66 H. sapiens 128 143447 4 41826 tcgtcagttcttagatcttg 67 H. sapiens 129 143448 4 41936 cctcctgcatccttgccttg 68 H. sapiens 130 143449 4 42120 tccttacactctgacatgaa 69 H. sapiens 131 143451 4 42280 cccgtggggctgagtctgtg 71 H. sapiens 132 143452 4 42302 cccattcggccaagctggat 72 H. sapiens 133 143453 4 42363 ccgagtacacgacccaggta 73 H. sapiens 134 143454 4 42401 ttgggcgcacccactggggc 74 H. sapiens 135 143455 4 42465 cctcacttcactgcattcca 75 H. sapiens 136 143456 4 42505 cttgctggggaagggcccac 76 H. sapiens 137 143457 4 42557 gccatctccctttgggaact 77 H. sapiens 138 143458 11 7852 atgtccctgtaaatgtggga 78 H. sapiens 139 143459 4 42651 caagaagaggagctgcctga 79 H. sapiens 140 143460 4 42682 tcttcctggtaatcctctgg 80 H. sapiens 141 143463 4 15976 cttgacctcaggaggcggag 83 H. sapiens 142 143467 4 35224 cgtggcccaggttagtgaca 87 H. sapiens 143 143469 4 40290 ctggccaacgtagtaaaacc 89 H. sapiens 144

[0197] As these “preferred target segments” have been found by experimentation to be open to, and accessible for, hybridization with the antisense compounds of the present invention, one of skill in the art will recognize or be able to ascertain, using no more than routine experimentation, further embodiments of the invention that encompass other compounds that specifically hybridize to these preferred target segments and consequently inhibit the expression of Notch3.

[0198] According to the present invention, antisense compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other short oligomeric compounds which hybridize to at least a portion of the target nucleic acid.

Example 16

[0199] Western Blot Analysis of Notch3 Protein Levels

[0200] Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to Notch3 is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

1 155 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1 tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence Antisense Oligonucleotide 2 gtgcgcgcga gcccgaaatc 20 3 20 DNA Artificial Sequence Antisense Oligonucleotide 3 atgcattctg cccccaagga 20 4 8091 DNA H. sapiens CDS (79)...(7044) 4 acgcggcgcg gaggctggcc cgggacgcgc ccggagccca gggaaggagg gaggagggga 60 gggtcgcggc cggccgcc atg ggg ccg ggg gcc cgt ggc cgc cgc cgc cgc 111 Met Gly Pro Gly Ala Arg Gly Arg Arg Arg Arg 1 5 10 cgt cgc ccg atg tcg ccg cca ccg cca ccg cca ccc gtg cgg gcg ctg 159 Arg Arg Pro Met Ser Pro Pro Pro Pro Pro Pro Pro Val Arg Ala Leu 15 20 25 ccc ctg ctg ctg ctg cta gcg ggg ccg ggg gct gca gcc ccc cct tgc 207 Pro Leu Leu Leu Leu Leu Ala Gly Pro Gly Ala Ala Ala Pro Pro Cys 30 35 40 ctg gac gga agc ccg tgt gca aat gga ggt cgt tgc acc cag ctg ccc 255 Leu Asp Gly Ser Pro Cys Ala Asn Gly Gly Arg Cys Thr Gln Leu Pro 45 50 55 tcc cgg gag gct gcc tgc ctg tgc ccg cct ggc tgg gtg ggt gag cgg 303 Ser Arg Glu Ala Ala Cys Leu Cys Pro Pro Gly Trp Val Gly Glu Arg 60 65 70 75 tgt cag ctg gag gac ccc tgt cac tca ggc ccc tgt gct ggc cgt ggt 351 Cys Gln Leu Glu Asp Pro Cys His Ser Gly Pro Cys Ala Gly Arg Gly 80 85 90 gtc tgc cag agt tca gtg gtg gct ggc acc gcc cga ttc tca tgc cgg 399 Val Cys Gln Ser Ser Val Val Ala Gly Thr Ala Arg Phe Ser Cys Arg 95 100 105 tgc ccc cgt ggc ttc cga ggc cct gac tgc tcc ctg cca gat ccc tgc 447 Cys Pro Arg Gly Phe Arg Gly Pro Asp Cys Ser Leu Pro Asp Pro Cys 110 115 120 ctc agc agc cct tgt gcc cac ggt gcc cgc tgc tca gtg ggg ccc gat 495 Leu Ser Ser Pro Cys Ala His Gly Ala Arg Cys Ser Val Gly Pro Asp 125 130 135 gga cgc ttc ctc tgc tcc tgc cca cct ggc tac cag ggc cgc agc tgc 543 Gly Arg Phe Leu Cys Ser Cys Pro Pro Gly Tyr Gln Gly Arg Ser Cys 140 145 150 155 cga agc gac gtg gat gag tgc cgg gtg ggt gag ccc tgc cgc cat ggt 591 Arg Ser Asp Val Asp Glu Cys Arg Val Gly Glu Pro Cys Arg His Gly 160 165 170 ggc acc tgc ctc aac aca cct ggc tcc ttc cgc tgc cag tgt cca gct 639 Gly Thr Cys Leu Asn Thr Pro Gly Ser Phe Arg Cys Gln Cys Pro Ala 175 180 185 ggc tac aca ggg cca cta tgt gag aac ccc gcg gtg ccc tgt gcg ccc 687 Gly Tyr Thr Gly Pro Leu Cys Glu Asn Pro Ala Val Pro Cys Ala Pro 190 195 200 tca cca tgc cgt aac ggg ggc acc tgc agg cag agt ggc gac ctc act 735 Ser Pro Cys Arg Asn Gly Gly Thr Cys Arg Gln Ser Gly Asp Leu Thr 205 210 215 tac gac tgt gcc tgt ctt cct ggg ttt gag ggt cag aat tgt gaa gtg 783 Tyr Asp Cys Ala Cys Leu Pro Gly Phe Glu Gly Gln Asn Cys Glu Val 220 225 230 235 aac gtg gac gac tgt cca gga cac cga tgt ctc aat ggg ggg aca tgc 831 Asn Val Asp Asp Cys Pro Gly His Arg Cys Leu Asn Gly Gly Thr Cys 240 245 250 gtg gat ggc gtc aac acc tat aac tgc cag tgc cct cct gag tgg aca 879 Val Asp Gly Val Asn Thr Tyr Asn Cys Gln Cys Pro Pro Glu Trp Thr 255 260 265 ggc cag ttc tgc acg gag gac gtg gat gag tgt cag ctg cag ccc aac 927 Gly Gln Phe Cys Thr Glu Asp Val Asp Glu Cys Gln Leu Gln Pro Asn 270 275 280 gcc tgc cac aat ggg ggt acc tgc ttc aac acg ctg ggt ggc cac agc 975 Ala Cys His Asn Gly Gly Thr Cys Phe Asn Thr Leu Gly Gly His Ser 285 290 295 tgc gtg tgt gtc aat ggc tgg aca ggt gag agc tgc agt cag aat atc 1023 Cys Val Cys Val Asn Gly Trp Thr Gly Glu Ser Cys Ser Gln Asn Ile 300 305 310 315 gat gac tgt gcc aca gcc gtg tgc ttc cat ggg gcc acc tgc cat gac 1071 Asp Asp Cys Ala Thr Ala Val Cys Phe His Gly Ala Thr Cys His Asp 320 325 330 cgc gtg gct tct ttc tac tgt gcc tgc ccc atg ggc aag act ggc ctc 1119 Arg Val Ala Ser Phe Tyr Cys Ala Cys Pro Met Gly Lys Thr Gly Leu 335 340 345 ctg tgt cac ctg gat gac gcc tgt gtc agc aac ccc tgc cac gag gat 1167 Leu Cys His Leu Asp Asp Ala Cys Val Ser Asn Pro Cys His Glu Asp 350 355 360 gct atc tgt gac aca aat ccg gtg aac ggc cgg gcc att tgc acc tgt 1215 Ala Ile Cys Asp Thr Asn Pro Val Asn Gly Arg Ala Ile Cys Thr Cys 365 370 375 cct ccc ggc ttc acg ggt ggg gca tgt gac cag gat gtg gac gag tgc 1263 Pro Pro Gly Phe Thr Gly Gly Ala Cys Asp Gln Asp Val Asp Glu Cys 380 385 390 395 tct atc ggc gcc aac ccc tgc gag cac ttg ggc agg tgc gtg aac acg 1311 Ser Ile Gly Ala Asn Pro Cys Glu His Leu Gly Arg Cys Val Asn Thr 400 405 410 cag ggc tcc ttc ctg tgc cag tgc ggt cgt ggc tac act gga cct cgc 1359 Gln Gly Ser Phe Leu Cys Gln Cys Gly Arg Gly Tyr Thr Gly Pro Arg 415 420 425 tgt gag acc gat gtc aac gag tgt ctg tcg ggg ccc tgc cga aac cag 1407 Cys Glu Thr Asp Val Asn Glu Cys Leu Ser Gly Pro Cys Arg Asn Gln 430 435 440 gcc acg tgc ctc gac cgc ata ggc cag ttc acc tgt atc tgt atg gca 1455 Ala Thr Cys Leu Asp Arg Ile Gly Gln Phe Thr Cys Ile Cys Met Ala 445 450 455 ggc ttc aca gga acc tat tgc gag gtg gac att gac gag tgt cag agt 1503 Gly Phe Thr Gly Thr Tyr Cys Glu Val Asp Ile Asp Glu Cys Gln Ser 460 465 470 475 agc ccc tgt gtc aac ggt ggg gtc tgc aag gac cga gtc aat ggc ttc 1551 Ser Pro Cys Val Asn Gly Gly Val Cys Lys Asp Arg Val Asn Gly Phe 480 485 490 agc tgc acc tgc ccc tcg ggc ttc agc ggc tcc acg tgt cag ctg gac 1599 Ser Cys Thr Cys Pro Ser Gly Phe Ser Gly Ser Thr Cys Gln Leu Asp 495 500 505 gtg gac gaa tgc gcc agc acg ccc tgc agg aat ggc gcc aaa tgc gtg 1647 Val Asp Glu Cys Ala Ser Thr Pro Cys Arg Asn Gly Ala Lys Cys Val 510 515 520 gac cag ccc gat ggc tac gag tgc cgc tgt gcc gag ggc ttt gag ggc 1695 Asp Gln Pro Asp Gly Tyr Glu Cys Arg Cys Ala Glu Gly Phe Glu Gly 525 530 535 acg ctg tgt gat cgc aac gtg gac gac tgc tcc cct gac cca tgc cac 1743 Thr Leu Cys Asp Arg Asn Val Asp Asp Cys Ser Pro Asp Pro Cys His 540 545 550 555 cat ggt cgc tgc gtg gat ggc atc gcc agc ttc tca tgt gcc tgt gct 1791 His Gly Arg Cys Val Asp Gly Ile Ala Ser Phe Ser Cys Ala Cys Ala 560 565 570 cct ggc tac acg ggc aca cgc tgc gag agc cag gtg gac gaa tgc cgc 1839 Pro Gly Tyr Thr Gly Thr Arg Cys Glu Ser Gln Val Asp Glu Cys Arg 575 580 585 agc cag ccc tgc cgc cat ggc ggc aaa tgc cta gac ctg gtg gac aag 1887 Ser Gln Pro Cys Arg His Gly Gly Lys Cys Leu Asp Leu Val Asp Lys 590 595 600 tac ctc tgc cgc tgc cct tct ggg acc aca ggt gtg aac tgc gaa gtg 1935 Tyr Leu Cys Arg Cys Pro Ser Gly Thr Thr Gly Val Asn Cys Glu Val 605 610 615 aac att gac gac tgt gcc agc aac ccc tgc acc ttt gga gtc tgc cgt 1983 Asn Ile Asp Asp Cys Ala Ser Asn Pro Cys Thr Phe Gly Val Cys Arg 620 625 630 635 gat ggc atc aac cgc tac gac tgt gtc tgc caa cct ggc ttc aca ggg 2031 Asp Gly Ile Asn Arg Tyr Asp Cys Val Cys Gln Pro Gly Phe Thr Gly 640 645 650 ccc ctt tgt aac gtg gag atc aat gag tgt gct tcc agc cca tgc ggc 2079 Pro Leu Cys Asn Val Glu Ile Asn Glu Cys Ala Ser Ser Pro Cys Gly 655 660 665 gag gga ggt tcc tgt gtg gat ggg gaa aat ggc ttc cgc tgc ctc tgc 2127 Glu Gly Gly Ser Cys Val Asp Gly Glu Asn Gly Phe Arg Cys Leu Cys 670 675 680 ccg cct ggc tcc ttg ccc cca ctc tgc ctc ccc ccg agc cat ccc tgt 2175 Pro Pro Gly Ser Leu Pro Pro Leu Cys Leu Pro Pro Ser His Pro Cys 685 690 695 gcc cat gag ccc tgc agt cac ggc atc tgc tat gat gca cct ggc ggg 2223 Ala His Glu Pro Cys Ser His Gly Ile Cys Tyr Asp Ala Pro Gly Gly 700 705 710 715 ttc cgc tgt gtg tgt gag cct ggc tgg agt ggc ccc cgc tgc agc cag 2271 Phe Arg Cys Val Cys Glu Pro Gly Trp Ser Gly Pro Arg Cys Ser Gln 720 725 730 agc ctg gcc cga gac gcc tgt gag tcc cag ccg tgc agg gcc ggt ggg 2319 Ser Leu Ala Arg Asp Ala Cys Glu Ser Gln Pro Cys Arg Ala Gly Gly 735 740 745 aca tgc agc agc gat gga atg ggt ttc cac tgc acc tgc ccg cct ggt 2367 Thr Cys Ser Ser Asp Gly Met Gly Phe His Cys Thr Cys Pro Pro Gly 750 755 760 gtc cag gga cgt cag tgt gaa ctc ctc tcc ccc tgc acc ccg aac ccc 2415 Val Gln Gly Arg Gln Cys Glu Leu Leu Ser Pro Cys Thr Pro Asn Pro 765 770 775 tgt gag cat ggg ggc cgc tgc gag tct gcc cct ggc cag ctg cct gtc 2463 Cys Glu His Gly Gly Arg Cys Glu Ser Ala Pro Gly Gln Leu Pro Val 780 785 790 795 tgc tcc tgc ccc cag ggc tgg caa ggc cca cga tgc cag cag gat gtg 2511 Cys Ser Cys Pro Gln Gly Trp Gln Gly Pro Arg Cys Gln Gln Asp Val 800 805 810 gac gag tgt gct ggc ccc gca ccc tgt ggc cct cat ggt atc tgc acc 2559 Asp Glu Cys Ala Gly Pro Ala Pro Cys Gly Pro His Gly Ile Cys Thr 815 820 825 aac ctg gca ggg agt ttc agc tgc acc tgc cat gga ggg tac act ggc 2607 Asn Leu Ala Gly Ser Phe Ser Cys Thr Cys His Gly Gly Tyr Thr Gly 830 835 840 cct tcc tgt gat cag gac atc aat gac tgt gac ccc aac cca tgc ctg 2655 Pro Ser Cys Asp Gln Asp Ile Asn Asp Cys Asp Pro Asn Pro Cys Leu 845 850 855 aac ggt ggc tcg tgc caa gac ggc gtg ggc tcc ttt tcc tgc tcc tgc 2703 Asn Gly Gly Ser Cys Gln Asp Gly Val Gly Ser Phe Ser Cys Ser Cys 860 865 870 875 ctc cct ggt ttc gcc ggc cca cga tgc gcc cgc gat gtg gat gag tgc 2751 Leu Pro Gly Phe Ala Gly Pro Arg Cys Ala Arg Asp Val Asp Glu Cys 880 885 890 ctg agc aac ccc tgc ggc ccg ggc acc tgt acc gac cac gtg gcc tcc 2799 Leu Ser Asn Pro Cys Gly Pro Gly Thr Cys Thr Asp His Val Ala Ser 895 900 905 ttc acc tgc acc tgc ccg ccg ggc tac gga ggc ttc cac tgc gaa cag 2847 Phe Thr Cys Thr Cys Pro Pro Gly Tyr Gly Gly Phe His Cys Glu Gln 910 915 920 gac ctg ccc gac tgc agc ccc agc tcc tgc ttc aat ggc ggg acc tgt 2895 Asp Leu Pro Asp Cys Ser Pro Ser Ser Cys Phe Asn Gly Gly Thr Cys 925 930 935 gtg gac ggc gtg aac tcg ttc agc tgc ctg tgc cgt ccc ggc tac aca 2943 Val Asp Gly Val Asn Ser Phe Ser Cys Leu Cys Arg Pro Gly Tyr Thr 940 945 950 955 gga gcc cac tgc caa cat gag gca gac ccc tgc ctc tcg cgg ccc tgc 2991 Gly Ala His Cys Gln His Glu Ala Asp Pro Cys Leu Ser Arg Pro Cys 960 965 970 cta cac ggg ggc gtc tgc agc gcc gcc cac cct ggc ttc cgc tgc acc 3039 Leu His Gly Gly Val Cys Ser Ala Ala His Pro Gly Phe Arg Cys Thr 975 980 985 tgc ctc gag agc ttc acg ggc ccg cag tgc cag acg ctg gtg gat tgg 3087 Cys Leu Glu Ser Phe Thr Gly Pro Gln Cys Gln Thr Leu Val Asp Trp 990 995 1000 tgc agc cgc cag cct tgt caa aac ggg ggt cgc tgc gtc cag act ggg 3135 Cys Ser Arg Gln Pro Cys Gln Asn Gly Gly Arg Cys Val Gln Thr Gly 1005 1010 1015 gcc tat tgc ctt tgt ccc cct gga tgg agc gga cgc ctc tgt gac atc 3183 Ala Tyr Cys Leu Cys Pro Pro Gly Trp Ser Gly Arg Leu Cys Asp Ile 1020 1025 1030 1035 cga agc ttg ccc tgc agg gag gcc gca gcc cag atc ggg gtg cgg ctg 3231 Arg Ser Leu Pro Cys Arg Glu Ala Ala Ala Gln Ile Gly Val Arg Leu 1040 1045 1050 gag cag ctg tgt cag gcg ggt ggg cag tgt gtg gat gaa gac agc tcc 3279 Glu Gln Leu Cys Gln Ala Gly Gly Gln Cys Val Asp Glu Asp Ser Ser 1055 1060 1065 cac tac tgc gtg tgc cca gag ggc cgt act ggt agc cac tgt gag cag 3327 His Tyr Cys Val Cys Pro Glu Gly Arg Thr Gly Ser His Cys Glu Gln 1070 1075 1080 gag gtg gac ccc tgc ttg gcc cag ccc tgc cag cat ggg ggg acc tgc 3375 Glu Val Asp Pro Cys Leu Ala Gln Pro Cys Gln His Gly Gly Thr Cys 1085 1090 1095 cgt ggc tat atg ggg ggc tac atg tgt gag tgt ctt cct ggc tac aat 3423 Arg Gly Tyr Met Gly Gly Tyr Met Cys Glu Cys Leu Pro Gly Tyr Asn 1100 1105 1110 1115 ggt gat aac tgt gag gac gac gtg gac gag tgt gcc tcc cag ccc tgc 3471 Gly Asp Asn Cys Glu Asp Asp Val Asp Glu Cys Ala Ser Gln Pro Cys 1120 1125 1130 cag cac ggg ggt tca tgc att gac ctc gtg gcc cgc tat ctc tgc tcc 3519 Gln His Gly Gly Ser Cys Ile Asp Leu Val Ala Arg Tyr Leu Cys Ser 1135 1140 1145 tgt ccc cca gga acg ctg ggg gtg ctc tgc gag att aat gag gat gac 3567 Cys Pro Pro Gly Thr Leu Gly Val Leu Cys Glu Ile Asn Glu Asp Asp 1150 1155 1160 tgc ggc cca ggc cca ccg ctg gac tca ggg ccc cgg tgc cta cac aat 3615 Cys Gly Pro Gly Pro Pro Leu Asp Ser Gly Pro Arg Cys Leu His Asn 1165 1170 1175 ggc acc tgc gtg gac ctg gtg ggt ggt ttc cgc tgc acc tgt ccc cca 3663 Gly Thr Cys Val Asp Leu Val Gly Gly Phe Arg Cys Thr Cys Pro Pro 1180 1185 1190 1195 gga tac act ggt ttg cgc tgc gag gca gac atc aat gag tgt cgc tca 3711 Gly Tyr Thr Gly Leu Arg Cys Glu Ala Asp Ile Asn Glu Cys Arg Ser 1200 1205 1210 ggt gcc tgc cac gcg gca cac acc cgg gac tgc ctg cag gac cca ggc 3759 Gly Ala Cys His Ala Ala His Thr Arg Asp Cys Leu Gln Asp Pro Gly 1215 1220 1225 gga ggt ttc cgt tgc ctt tgt cat gct ggc ttc tca ggt cct cgc tgt 3807 Gly Gly Phe Arg Cys Leu Cys His Ala Gly Phe Ser Gly Pro Arg Cys 1230 1235 1240 cag act gtc ctg tct ccc tgc gag tcc cag cca tgc cag cat gga ggc 3855 Gln Thr Val Leu Ser Pro Cys Glu Ser Gln Pro Cys Gln His Gly Gly 1245 1250 1255 cag tgc cgt cct agc ccg ggt cct ggg ggt ggg ctg acc ttc acc tgt 3903 Gln Cys Arg Pro Ser Pro Gly Pro Gly Gly Gly Leu Thr Phe Thr Cys 1260 1265 1270 1275 cac tgt gcc cag ccg ttc tgg ggt ccg cgt tgc gag cgg gtg gcg cgc 3951 His Cys Ala Gln Pro Phe Trp Gly Pro Arg Cys Glu Arg Val Ala Arg 1280 1285 1290 tcc tgc cgg gag ctg cag tgc ccg gtg ggc gtc cca tgc cag cag acg 3999 Ser Cys Arg Glu Leu Gln Cys Pro Val Gly Val Pro Cys Gln Gln Thr 1295 1300 1305 ccc cgc ggg ccg cgc tgc gcc tgc ccc cca ggg ttg tcg gga ccc tcc 4047 Pro Arg Gly Pro Arg Cys Ala Cys Pro Pro Gly Leu Ser Gly Pro Ser 1310 1315 1320 tgc cgc agc ttc ccg ggg tcg ccg ccg ggg gcc agc aac gcc agc tgc 4095 Cys Arg Ser Phe Pro Gly Ser Pro Pro Gly Ala Ser Asn Ala Ser Cys 1325 1330 1335 gcg gcc gcc ccc tgt ctc cac ggg ggc tcc tgc cgc ccc gcg ccg ctc 4143 Ala Ala Ala Pro Cys Leu His Gly Gly Ser Cys Arg Pro Ala Pro Leu 1340 1345 1350 1355 gcg ccc ttc ttc cgc tgc gct tgc gcg cag ggc tgg acc ggg ccg cgc 4191 Ala Pro Phe Phe Arg Cys Ala Cys Ala Gln Gly Trp Thr Gly Pro Arg 1360 1365 1370 tgc gag gcg ccc gcc gcg gca ccc gag gtc tcg gag gag ccg cgg tgc 4239 Cys Glu Ala Pro Ala Ala Ala Pro Glu Val Ser Glu Glu Pro Arg Cys 1375 1380 1385 ccg cgc gcc gcc tgc cag gcc aag cgc ggg gac cag cgc tgc gac cgc 4287 Pro Arg Ala Ala Cys Gln Ala Lys Arg Gly Asp Gln Arg Cys Asp Arg 1390 1395 1400 gag tgc aac agc cca ggc tgc ggc tgg gac ggc ggc gac tgc tcg ctg 4335 Glu Cys Asn Ser Pro Gly Cys Gly Trp Asp Gly Gly Asp Cys Ser Leu 1405 1410 1415 agc gtg ggc gac ccc tgg cgg caa tgc gag gcg ctg cag tgc tgg cgc 4383 Ser Val Gly Asp Pro Trp Arg Gln Cys Glu Ala Leu Gln Cys Trp Arg 1420 1425 1430 1435 ctc ttc aac aac agc cgc tgc gac ccc gcc tgc agc tcg ccc gcc tgc 4431 Leu Phe Asn Asn Ser Arg Cys Asp Pro Ala Cys Ser Ser Pro Ala Cys 1440 1445 1450 ctc tac gac aac ttc gac tgc cac gcc ggt ggc cgc gag cgc act tgc 4479 Leu Tyr Asp Asn Phe Asp Cys His Ala Gly Gly Arg Glu Arg Thr Cys 1455 1460 1465 aac ccg gtg tac gag aag tac tgc gcc gac cac ttt gcc gac ggc cgc 4527 Asn Pro Val Tyr Glu Lys Tyr Cys Ala Asp His Phe Ala Asp Gly Arg 1470 1475 1480 tgc gac cag ggc tgc aac acg gag gag tgc ggc tgg gat ggg ctg gat 4575 Cys Asp Gln Gly Cys Asn Thr Glu Glu Cys Gly Trp Asp Gly Leu Asp 1485 1490 1495 tgt gcc agc gag gtg ccg gcc ctg ctg gcc cgc ggc gtg ctg gtg ctc 4623 Cys Ala Ser Glu Val Pro Ala Leu Leu Ala Arg Gly Val Leu Val Leu 1500 1505 1510 1515 aca gtg ctg ctg ccg ccg gag gag cta ctg cgt tcc agc gcc gac ttt 4671 Thr Val Leu Leu Pro Pro Glu Glu Leu Leu Arg Ser Ser Ala Asp Phe 1520 1525 1530 ctg cag cgg ctc agc gcc atc ctg cgc acc tcg ctg cgc ttc cgc ctg 4719 Leu Gln Arg Leu Ser Ala Ile Leu Arg Thr Ser Leu Arg Phe Arg Leu 1535 1540 1545 gac gcg cac ggc cag gcc atg gtc ttc cct tac cac cgg cct agt cct 4767 Asp Ala His Gly Gln Ala Met Val Phe Pro Tyr His Arg Pro Ser Pro 1550 1555 1560 ggc tcc gaa ccc cgg gcc cgt cgg gag ctg gcc ccc gag gtg atc ggc 4815 Gly Ser Glu Pro Arg Ala Arg Arg Glu Leu Ala Pro Glu Val Ile Gly 1565 1570 1575 tcg gta gta atg ctg gag att gac aac cgg ctc tgc ctg cag tcg cct 4863 Ser Val Val Met Leu Glu Ile Asp Asn Arg Leu Cys Leu Gln Ser Pro 1580 1585 1590 1595 gag aat gat cac tgc ttc ccc gat gcc cag agc gcc gct gac tac ctg 4911 Glu Asn Asp His Cys Phe Pro Asp Ala Gln Ser Ala Ala Asp Tyr Leu 1600 1605 1610 gga gcg ttg tca gcg gtg gag cgc ctg gac ttc ccg tac cca ctg cgg 4959 Gly Ala Leu Ser Ala Val Glu Arg Leu Asp Phe Pro Tyr Pro Leu Arg 1615 1620 1625 gac gtg cgg ggg gag ccg ctg gag cct cca gaa ccc agc gtc ccg ctg 5007 Asp Val Arg Gly Glu Pro Leu Glu Pro Pro Glu Pro Ser Val Pro Leu 1630 1635 1640 ctg cca ctg cta gtg gcg ggc gct gtc ttg ctg ctg gtc att ctc gtc 5055 Leu Pro Leu Leu Val Ala Gly Ala Val Leu Leu Leu Val Ile Leu Val 1645 1650 1655 ctg ggt gtc atg gtg gcc cgg cgc aag cgc gag cac agc acc ctc tgg 5103 Leu Gly Val Met Val Ala Arg Arg Lys Arg Glu His Ser Thr Leu Trp 1660 1665 1670 1675 ttc cct gag ggc ttc tca ctg cac aag gac gtg gcc tct ggt cac aag 5151 Phe Pro Glu Gly Phe Ser Leu His Lys Asp Val Ala Ser Gly His Lys 1680 1685 1690 ggc cgg cgg gaa ccc gtg ggc cag gac gcg ctg ggc atg aag aac atg 5199 Gly Arg Arg Glu Pro Val Gly Gln Asp Ala Leu Gly Met Lys Asn Met 1695 1700 1705 gcc aag ggt gag agc ctg atg ggg gag gtg gcc aca gac tgg atg gac 5247 Ala Lys Gly Glu Ser Leu Met Gly Glu Val Ala Thr Asp Trp Met Asp 1710 1715 1720 aca gag tgc cca gag gcc aag cgg cta aag gta gag gag cca ggc atg 5295 Thr Glu Cys Pro Glu Ala Lys Arg Leu Lys Val Glu Glu Pro Gly Met 1725 1730 1735 ggg gct gag gag gct gtg gat tgc cgt cag tgg act caa cac cat ctg 5343 Gly Ala Glu Glu Ala Val Asp Cys Arg Gln Trp Thr Gln His His Leu 1740 1745 1750 1755 gtt gct gct gac atc cgc gtg gca cca gcc atg gca ctg aca cca cca 5391 Val Ala Ala Asp Ile Arg Val Ala Pro Ala Met Ala Leu Thr Pro Pro 1760 1765 1770 cag ggc gac gca gat gct gat ggc atg gat gtc aat gtg cgt ggc cca 5439 Gln Gly Asp Ala Asp Ala Asp Gly Met Asp Val Asn Val Arg Gly Pro 1775 1780 1785 gat ggc ttc acc ccg cta atg ctg gct tcc ttc tgt ggg ggg gct ctg 5487 Asp Gly Phe Thr Pro Leu Met Leu Ala Ser Phe Cys Gly Gly Ala Leu 1790 1795 1800 gag cca atg cca act gaa gag gat gag gca gat gac aca tca gct agc 5535 Glu Pro Met Pro Thr Glu Glu Asp Glu Ala Asp Asp Thr Ser Ala Ser 1805 1810 1815 atc atc tcc gac ctg atc tgc cag ggg gct cag ctt ggg gca cgg act 5583 Ile Ile Ser Asp Leu Ile Cys Gln Gly Ala Gln Leu Gly Ala Arg Thr 1820 1825 1830 1835 gac cgt act ggc gag act gct ttg cac ctg gct gcc cgt tat gcc cgt 5631 Asp Arg Thr Gly Glu Thr Ala Leu His Leu Ala Ala Arg Tyr Ala Arg 1840 1845 1850 gct gat gca gcc aag cgg ctg ctg gat gct ggg gca gac acc aat gcc 5679 Ala Asp Ala Ala Lys Arg Leu Leu Asp Ala Gly Ala Asp Thr Asn Ala 1855 1860 1865 cag gac cac tca ggc cgc act ccc ctg cac aca gct gtc aca gcc gat 5727 Gln Asp His Ser Gly Arg Thr Pro Leu His Thr Ala Val Thr Ala Asp 1870 1875 1880 gcc cag ggt gtc ttc cag att ctc atc cga aac cgc tct aca gac ttg 5775 Ala Gln Gly Val Phe Gln Ile Leu Ile Arg Asn Arg Ser Thr Asp Leu 1885 1890 1895 gat gcc cgc atg gca gat ggc tca acg gca ctg atc ctg gcg gcc cgc 5823 Asp Ala Arg Met Ala Asp Gly Ser Thr Ala Leu Ile Leu Ala Ala Arg 1900 1905 1910 1915 ctg gca gta gag ggc atg gtg gaa gag ctc atc gcc agc cat gct gat 5871 Leu Ala Val Glu Gly Met Val Glu Glu Leu Ile Ala Ser His Ala Asp 1920 1925 1930 gtc aat gct gtg gat gag ctt ggg aaa tca gcc tta cac tgg gct gcg 5919 Val Asn Ala Val Asp Glu Leu Gly Lys Ser Ala Leu His Trp Ala Ala 1935 1940 1945 gct gtg aac aac gtg gaa gcc act ttg gcc ctg ctc aaa aat gga gcc 5967 Ala Val Asn Asn Val Glu Ala Thr Leu Ala Leu Leu Lys Asn Gly Ala 1950 1955 1960 aat aag gac atg cag gat agc aag gag gag acc ccc cta ttc ctg gcc 6015 Asn Lys Asp Met Gln Asp Ser Lys Glu Glu Thr Pro Leu Phe Leu Ala 1965 1970 1975 gcc cgc gag ggc agc tat gag gct gcc aag ctg ctg ttg gac cac ttt 6063 Ala Arg Glu Gly Ser Tyr Glu Ala Ala Lys Leu Leu Leu Asp His Phe 1980 1985 1990 1995 gcc aac cgt gag atc acc gac cac ctg gac agg ctg ccg cgg gac gta 6111 Ala Asn Arg Glu Ile Thr Asp His Leu Asp Arg Leu Pro Arg Asp Val 2000 2005 2010 gcc cag gag aga ctg cac cag gac atc gtg cgc ttg ctg gat caa ccc 6159 Ala Gln Glu Arg Leu His Gln Asp Ile Val Arg Leu Leu Asp Gln Pro 2015 2020 2025 agt ggg ccc cgc agc ccc ccc ggt ccc cac ggc ctg ggg cct ctg ctc 6207 Ser Gly Pro Arg Ser Pro Pro Gly Pro His Gly Leu Gly Pro Leu Leu 2030 2035 2040 tgt cct cca ggg gcc ttc ctc cct ggc ctc aaa gcg gca cag tcg ggg 6255 Cys Pro Pro Gly Ala Phe Leu Pro Gly Leu Lys Ala Ala Gln Ser Gly 2045 2050 2055 tcc aag aag agc agg agg ccc ccc ggg aag gcg ggg ctg ggg ccg cag 6303 Ser Lys Lys Ser Arg Arg Pro Pro Gly Lys Ala Gly Leu Gly Pro Gln 2060 2065 2070 2075 ggg ccc cgg ggg cgg ggc aag aag ctg acg ctg gcc tgc ccg ggc ccc 6351 Gly Pro Arg Gly Arg Gly Lys Lys Leu Thr Leu Ala Cys Pro Gly Pro 2080 2085 2090 ctg gct gac agc tcg gtc acg ctg tcg ccc gtg gac tcg ctg gac tcc 6399 Leu Ala Asp Ser Ser Val Thr Leu Ser Pro Val Asp Ser Leu Asp Ser 2095 2100 2105 ccg cgg cct ttc ggt ggg ccc cct gct tcc cct ggt ggc ttc ccc ctt 6447 Pro Arg Pro Phe Gly Gly Pro Pro Ala Ser Pro Gly Gly Phe Pro Leu 2110 2115 2120 gag ggg ccc tat gca gct gcc act gcc act gca gtg tct ctg gca cag 6495 Glu Gly Pro Tyr Ala Ala Ala Thr Ala Thr Ala Val Ser Leu Ala Gln 2125 2130 2135 ctt ggt ggc cca ggc cgg gca ggt cta ggg cgc cag ccc cct gga gga 6543 Leu Gly Gly Pro Gly Arg Ala Gly Leu Gly Arg Gln Pro Pro Gly Gly 2140 2145 2150 2155 tgt gta ctc agc ctg ggc ctg ctg aac cct gtg gct gtg ccc ctc gat 6591 Cys Val Leu Ser Leu Gly Leu Leu Asn Pro Val Ala Val Pro Leu Asp 2160 2165 2170 tgg gcc cgg ctg ccc cca cct gcc cct cca ggc ccc tcg ttc ctg ctg 6639 Trp Ala Arg Leu Pro Pro Pro Ala Pro Pro Gly Pro Ser Phe Leu Leu 2175 2180 2185 cca ctg gcg ccg gga ccc cag ctg ctc aac cca ggg acc ccc gtc tcc 6687 Pro Leu Ala Pro Gly Pro Gln Leu Leu Asn Pro Gly Thr Pro Val Ser 2190 2195 2200 ccg cag gag cgg ccc ccg cct tac ctg gca gtc cca gga cat ggc gag 6735 Pro Gln Glu Arg Pro Pro Pro Tyr Leu Ala Val Pro Gly His Gly Glu 2205 2210 2215 gag tac ccg gtg gct ggg gca cac agc agc ccc cca aag gcc cgc ttc 6783 Glu Tyr Pro Val Ala Gly Ala His Ser Ser Pro Pro Lys Ala Arg Phe 2220 2225 2230 2235 ctg cgg gtt ccc agt gag cac cct tac ctg acc cca tcc ccc gaa tcc 6831 Leu Arg Val Pro Ser Glu His Pro Tyr Leu Thr Pro Ser Pro Glu Ser 2240 2245 2250 cct gag cac tgg gcc agc ccc tca cct ccc tcc ctc tca gac tgg tcc 6879 Pro Glu His Trp Ala Ser Pro Ser Pro Pro Ser Leu Ser Asp Trp Ser 2255 2260 2265 gaa tcc acg cct agc cca gcc act gcc act ggg gcc atg gcc acc acc 6927 Glu Ser Thr Pro Ser Pro Ala Thr Ala Thr Gly Ala Met Ala Thr Thr 2270 2275 2280 act ggg gca ctg cct gcc cag cca ctt ccc ttg tct gtt ccc agc tcc 6975 Thr Gly Ala Leu Pro Ala Gln Pro Leu Pro Leu Ser Val Pro Ser Ser 2285 2290 2295 ctt gct cag gcc cag acc cag ctg ggg ccc cag ccg gaa gtt acc ccc 7023 Leu Ala Gln Ala Gln Thr Gln Leu Gly Pro Gln Pro Glu Val Thr Pro 2300 2305 2310 2315 aag agg caa gtg ttg gcc tga gacgctcgtc agttcttaga tcttgggggc 7074 Lys Arg Gln Val Leu Ala 2320 ctaaagagac ccccgtcctg cctcctttct ttctctgtct cttccttcct tttagtcttt 7134 ttcatcctct tctctttcca ccaaccctcc tgcatccttg ccttgcagcg tgaccgagat 7194 aggtcatcag cccagggctt cagtcttcct ttatttataa tgggtggggg ctaccaccca 7254 ccctctcagt cttgtgaaga gtctgggacc tccttcttcc ccacttctct cttccctcat 7314 tcctttctct ctccttctgg cctctcattt ccttacactc tgacatgaat gaattattat 7374 tatttttctt tttctttttt tttttacatt ttgtatagaa acaaattcat ttaaacaaac 7434 ttattattat tattttttac aaaatatata tatggagatg ctccctcccc ctgtgaaccc 7494 cccagtgccc ccgtggggct gagtctgtgg gcccattcgg ccaagctgga ttctgtgtac 7554 ctagtacaca ggcatgactg ggatcccgtg taccgagtac acgacccagg tatgtaccaa 7614 gtaggcaccc ttgggcgcac ccactggggc caggggtcgg gggagtgttg ggagcctcct 7674 ccccacccca cctccctcac ttcactgcat tccagattgg acatgttcca tagccttgct 7734 ggggaagggc ccactgccaa ctccctctgc cccagcccca cccttggcca tctccctttg 7794 ggaactaggg ggctgctggt gggaaatggg agccagggca gatgtatgca ttcctttatg 7854 tccctgtaaa tgtgggacta caagaagagg agctgcctga gtggtacttt ctcttcctgg 7914 taatcctctg gcccagcctt atggcagaat agaggtattt ttaggctatt tttgtaatat 7974 ggcttctggt caaaatccct gtgtagctga attcccaagc cctgcattgt acagcccccc 8034 actcccctca ccacctaata aaggaatagt taacactcaa aaaaaaaaaa aaaaaaa 8091 5 18 DNA Artificial Sequence PCR Primer 5 tcaccatgcc gtaacggg 18 6 19 DNA Artificial Sequence PCR Primer 6 tcggtgtcct ggacagtcg 19 7 28 DNA Artificial Sequence PCR Probe 7 cttcctgggt ttgagggtca gaattgtg 28 8 19 DNA Artificial Sequence PCR Primer 8 gaaggtgaag gtcggagtc 19 9 20 DNA Artificial Sequence PCR Primer 9 gaagatggtg atgggatttc 20 10 20 DNA Artificial Sequence PCR Probe 10 caagcttccc gttctcagcc 20 11 44348 DNA Homo sapiens 11 gtggctcacg cctgtaatcc caacactttg ggaggctgag gcaggcagat cacgaggtca 60 ggagatcgag accatcctgg ctgacacggt gaaaccccgt ctctactaaa aatacaaaaa 120 aaaaattagc agagcatggt gccagttgcc tgtagtccca gctactcgga cggctgaggc 180 aggagaatgg tgtgaaccca ggaggcggaa cttgcagtga gcggagatca tgccactgca 240 cttcagcctg ggcaacagag cgagactccg tctcaaaaac aaacaaacaa gtaaacaaaa 300 acccagaaaa aattagccac gtatggtggc gcacacctct aatcccagct acttggcaga 360 ttgtgggggt gggggctgag gaaggagaat cgcttgaacc tgggaggtag agcttgcagt 420 gagccaagat agggacactg cactccagcc tgggtgacag agcgacacgc tgtctcaaaa 480 aaaaaaaaaa aaagaaaaga gaattaatca aattgagcca gataggctga ggcccaaagc 540 tgtgtggggt atgggggtgg gactaggggg ctggggtgac cctttgagag tcacagagga 600 agtgggttgc tttctgggag ggctgagagc aagaagagtt tgtgtgtgca tgtgtgaaca 660 cacacacatg catgtgactc ctagtacgtg tctgaggtct gaggctgcaa atgtagtcct 720 aggctcctgg cctggctgag tgagttttag cggttttttg tttgtttgtt ttgtttgaga 780 cagggtctta ctctgttgcc caggctggag cgcagtggta cgatttcggc taagtgcaac 840 ctcagcctcc tgggttgaag cgattttcct gcttcagcct ctcgaatagc tgggactaca 900 ggcgtgcgcc accacgctca gctaattttt gtattttcag tagagtccgg gtttcaccat 960 gttggccagg ctggtctcca actcttgacc tcaagtgatc catccacctc tgcctctcaa 1020 agtgctggga ttacaggccg gagccaccgc gcccggtccg acttttaggt ttgtgaatgt 1080 tttagatcag agtcctgggg aagcttggtg catgggcttg catttgtgcg tccgtggctg 1140 tgggtccatg agcctctcag gacgtgactg gcctcagttt ccagagtttc tgggaggctg 1200 tgttttttgt cccggctcca gaggtgtccg gctctgggtg tgtactgggg gatggggatg 1260 gggtgcgtgg gcgttcacga ggttgggtgt gcccgccact ccgggttctg cccgcgtctc 1320 actgcatgct cggcctgggt ttccgagggt ccgcgcgtcc caggctgtgc gggtggaggg 1380 tgggcaggga ccccgggagg ccgggcgggg ggcgggggcg cgctgggccg gccccggggc 1440 ggggcgagcc ttcgagggct gggggcgggg cggcccggcc gcctcacttc ggcgaagttg 1500 gcggcgcgga ggctggcccg ggacgcgccc ggagcccagg gaaggaggga ggaggggagg 1560 gtcgcggccg gccgccatgg ggccgggggc ccgtggccgc cgccgccgcc gtcgcccgat 1620 gtcgccgcca ccgccaccgc cacccgtgcg ggcgctgccc ctgctgctgc tgctagcggg 1680 gccgggggct gcaggtgagg ggccgggacc tggcggatgg gacgagggcg gcagaggggg 1740 agtgcaagaa cccccaaggc cggggctggc gggggttcat gggaggcagg aaccagggtc 1800 ggggaagggg cgcaggagcc ccgggcttca tgccagtcct ggaggaccca gagattcaga 1860 atggggagga ccccagaggc ccaaggaaca gggacccttg agcgattaga gctgaagatg 1920 aagggaccca ggagtccgag actgggagct cgaggtgcgg ggatcaggga ctcgaggtgg 1980 gggggtgcgt acagagttcg ggactcgtcc ccatccaact cacgcctgga gtcctgggta 2040 ggttatgatt gggggcccag gtacttctag gccggggacc tctcgcacaa aagccccccc 2100 accccgcccc cgacaccccg ggcgggctgg gccaggcggg gggtggggag ggggcgcgaa 2160 gttctgggag ctctgaactc ggagaaaact tcccaggccg gcgcgcagca agacccggag 2220 ccggattccg agccggagcc tcggcggcgc gcgcgccccc tcccccgccc gcagcccgcc 2280 tctctcctct ggccgcgggg acccggaggc cctgggaccc cgcccctgcg cggggagggg 2340 aaggggcgag ggcccacgtg ctccccttcg gctccagcgc cccctccccg cggccagagc 2400 ccctccccag ccggccaggg gcccccgccc ctcctcctct cctccctccc ctcccccgct 2460 cgggacaatg gccgcgccgt ctagacaccc cctccctccg gccggcctcg cgctttcttt 2520 gccagacaaa gcgggacccg cggctgggcc ggggaggggg ctgcgggggc acccccctca 2580 ccgctacggg aggcctggtg ggcgggggag gggcgcgggc caaggcccct ggccaggggt 2640 cccagacgcc agtgtggggc ttggcgctgg gcggggtggg ggtttcggga gtgaacggcc 2700 tccccagccc agccccgggg cccggacggg gcaggaccag gcaggagccg ccgcctccgc 2760 cggaccagcg gcgcacacac atggcctgtg acacactcgc tggcacacat agctctaggt 2820 cacgtaacag agatgcaaac ctgcacacac acagcacact cgcagacttg cacacctggc 2880 tcaggaaaga cacacccagc tgcacgcaca cagctcacac attgacatac atacacacac 2940 aaatatgatc acacaggtgt ggcacacacg ctgcctgcac acactcagac agcacataga 3000 tgaagactca aggacatgct tacacccagc tcacacatgc agatggacag acacagccaa 3060 cccatacaca gacacagcgg tgcacacaca ggtcacacca acacacagct gtacacccat 3120 ttacacggtt cacatacaca gacgcacaac agacacaacc tgcatacaga cagcacacac 3180 atctgtgcat tcccgtatgg gtgtgcagct catacacaca cgggcacacc cacgctgaca 3240 tgcatgtaca gatagactca catgtagctg caactgagac acaatagatg catgcccaaa 3300 tccacataca gacacacaag cccgtgcacg cacacacatg tggctcacat tgcctacagc 3360 tcagggtggc ccatagccca tgttatggga cccacagtgt gagttcacga acacccacag 3420 gtcctggtgt gggtgatggt gtccagtcgg cttgtcctgt gaggaagggg catatactat 3480 gtaggcccat gctcaggttc agctcgacag attctgtaac tcaccctctt gttttctatt 3540 tctttctgtc cttctctctc tctttttttt ttttgagaca gtctcactct gttgccaggc 3600 tggcatgcag tggcgcaatc tcgggtcact gcaacctccg cttcccaggt tcaagcgatt 3660 ctcctgcctc agcctcccga gtagctggga ctacaggcgt gcgccaccac acccagctaa 3720 tttttttttg gagatggagt ttggctcttg ttgcccaggg tggagtgcaa tggcgtgatc 3780 tcggctcact ggaacctctg gctcctgggt tcaagcgatt ctcctgcctc agcctcctga 3840 gtagctagga ttacaggcac ctgctaccat gcccagctaa gttttatatt tttagaacag 3900 actgggtttc actgtgttgg ccaggctggt ctcgaactcc tgacctcagg tgatccacca 3960 gcctcggcct cccaaagtgt tgggattaga ggcgtgagcc accatgcctg gccgctttct 4020 atttctttct gtccttctct gtcttctcct ggtctatctg cttctagttc attctcacgt 4080 ctttgtgttt ttctcgatct tccttcctct ctccatcacc ctctctctga caagagcatg 4140 catggacaca cgcactgtgt ccccacacct gatcgcatgc acacagttct ccactagctc 4200 agacccagcc acaccctgga cccttctggc aggacagtta gactatgcca caccctagag 4260 ggacacagcc cttagggatg gcgatgttgg acagggcagc acatgccttg tgccagttgc 4320 ccctcccttc aacacacacc ctattccagc aacgcccagc gctggctgct tctggagatg 4380 ctgtgctcac tcgtgggtac atgccttggt ccctctccca agagcaccac gcaggcctgc 4440 ctggctggag tagatgccca tcagggaaag gaagacccgt ggccctagga tcccctgccc 4500 cttccccttc tattacccct gaacctgggc atgaagcccc agacctccct gtaacactca 4560 gcacccttac ttcctgtctc agcacacccc attctgcaca tcttagctct ggcagcttcc 4620 cgagccccca catctggcat aagacagccc ctcctcctgc cttcccctgc tttgtggttc 4680 ccagggctcg agctggcagg gacagctgca gcctcaacag ctggggctgg ggcggggggg 4740 gggggggcgt gggaactgtg gatgggggga ctccctgcac ccgcagagac gcccccatcc 4800 ccatgcagct gttgcctggt ggagggaggg agggggtttg tcacttgggc ctggggttcc 4860 tgggcacctg gctgatcctc caccttcctt cacccccaca cagccccccc ttgcctggac 4920 ggaagcccgt gtgcaaatgg aggtcgttgc acccagctgc cctcccggga ggctgcctgc 4980 ctgtgagtgc ctggctcaga gccaccagtg ggccctgtgt gtgggggcgg gggggagggg 5040 cgatttgtct tctccctctg cctctgtgtt caccatggat gtctctacca cttcctacat 5100 gtgtgtggct tgggcaacct ctcgtgtttc cccattggaa actgagcagg ggctctgcac 5160 tcttcttggc atgggttcaa gtcccacatc atgttgctga ccggctagga attaaactct 5220 ttacatctca gtgtcatggt ctgcaaaatg ggcccagtaa tcccagcttc acagttcttg 5280 gccacctgag gtcacatgta aagcccccca tgaggtaggt attattcaat caacaaacta 5340 tcagggacca atcgcagtgg ctcacaccct ccacacccct cccatctctc aacatttagt 5400 ggagggggac ataggaggcc tataatacag tcccacactg ccacttactt gcagtgtgac 5460 tttgggctag tcactttccc tctctgatca tcattgcacc tgtgaaatag ggaatgcttc 5520 actggcgact gtgaagggac ttaacaattt agttgttaga tatcacaagg aaaggcaaga 5580 ggttgcaggg agcagagcag aggctgggaa ggagctgagc agagacagaa agagctcagc 5640 ttaaattctg gggtctggga caccttgagc attcaagctt tttgagcctc agtttttgca 5700 tctggaaaat ggggcaataa taactcctgt atgcactcaa catacattaa gtgctcagaa 5760 tatagtatgt gctcaacata ggcattatta taaataataa tggtagggag gcaggaaaca 5820 ggtgctctaa ggagaagtct ctcaaggtta tgaggaaatg cttggctcct ttcatgctat 5880 cattaataac agcagttcac gcagtagcca ttgattgact cattaatgta ttcatttatt 5940 cagcatcatg tgtgccaagt actgggtcag gcccaagctg ggtgggtctg gggtgatttt 6000 agaccaagaa gagccgcttt gaagttacac ctacatttat tttggctgga tctcccactc 6060 gggctcacct ttgtggaggg ctgagcctga ctgagagggt ttcctggggc tggggggtct 6120 gcgaaggtgg ggcgggatgg ggaaaagtgt gaccttacct ggagccacac acctgggagg 6180 gcgagcggtg gggccgggcg cctgcgcagt ggagctccgc gcctggaata ctgccgacag 6240 gtgaatgagc ccgcggctgc cccgcccttc gacaggtgaa tcaccggcgc gcgcggcgcc 6300 cggagcccgg atcgcccgga gtggagcggg ctcagtcctc cgagttgggc tgtgggaacc 6360 actccctaca cgccctccac cccccacgag tctctttcac ggtctcaaat actcaagtcc 6420 tttcaaggat gcccccaagc tgtcacccca cccatggatc ccccaagacc cccccaccgg 6480 cagcagacgt ttccatagac ctcgtcccca tctcctagtc ctcgcctcac ccgccgtgcc 6540 ccccctaact aggtctgcgc tcccccctcc gtctccccca aagcttaggc cgtggggcgg 6600 ggcgcgggct ggggctggaa ccggcccgac cggtcggcgg gggcgcggga cgcagagcgt 6660 gggaacccgc ccggggcgtc gggagggggc ccgcgcgggt cgcgccctgc ctggcggtgg 6720 gaccagctat cctcggcgcc cagcgcagcg cgccccctcc cgacgcgcgg tcggggccgc 6780 agtggtcgcc ctgcgggcct tggaggaggg gacgggagct gtgccctccc ctcccaacgc 6840 cacccgcacc cttgcttgct cggccgtgcc ccgacctgtt tcgctggggg ccggggtggg 6900 ggggatcttg cgggtgacgc taaggactga gtcagccgct tgttgagttc agtctcaggc 6960 gtctgggaca agccggaggg aggacaaccg cgccaggggc ggagggtggg gggatagagg 7020 ggggtgaggg ttggcagcgt cggggggcgg agcttggact ctctggcttc tctaagcccc 7080 tccctctgac cccgctagtg ccccttggag atttccagtc ttaagaccaa cccctcccag 7140 ttccaattcc tcacactcct tggtgggctt ggggaggggg ctgcaggttg aaggaccacc 7200 cccccaagat gagggtacca acagtggtgt cagtacccca accctccacc ctcccatccc 7260 tgcataagag gctatataag tcccagagaa gggacttgag ggtttgtggg agccctgctg 7320 tctgtccctg tgagtgagag ttgctcattt ccgcgtggga tgctgtgggt tggtgtcttg 7380 agtgctggcc aggccatggc gtccaattgg tgtgtccctc tgtgtgtgta gctgtggttt 7440 ctgggcctgt ctgtgtgtgc ctgcgtggtt gtgtcctggg gtctgagact tctttttttc 7500 cttttccttt tttttttttt ttttgagatc gagtatcgct cttgttgccc aggctggagt 7560 gcagtggctc gatcttggct cactgcagcc tctatctcct gggttcaagc gattttcctg 7620 cctcagcctc cggagtagct gggattacag gcatgtgcca ccacacttgg ctgatttttt 7680 gtgtttttag tagagacgga gtttcttcat gttggtcagg ctggtctcga actcttgatg 7740 tcaggtgatc tacctgcctc ggtctcccaa agtgttggga ttacaggcgt gagccaccgc 7800 gcttggacat gggtctgaga cttttcattt gctgttccct ctgcctggaa tgccgttccc 7860 cagaaagccc catggccccc tccttacctt catatgtctg tgaaaataga attccacctc 7920 cttgacccca tcattctatc ccccttaacc ctgtggttta gcttcctacg gctgctctaa 7980 caaattacca taaactgggt agcttgaaca atagtaattt attctcttga agttctggag 8040 gttggaagtc tgaaatcaag atatcggcag ggctgagcct ctccatgggg acccagggga 8100 gaactgactc ttgcagcttc tggtggctcc agacgtttct cagctagtgg ctgtctcact 8160 ccagtccctg tctctgtctt cttctcttcc ttctcccacc caaatctcct tttggctgcc 8220 tctctctttt tttgagacag agtcttgctc tgtcacccag gctggagtgc agtggtgcaa 8280 tcttagttca ctgcagcctt caactcccag gctgaagcga tcctcccacc tcagcctccc 8340 aagaagctgg gactacaggt gtgagccacc acggccagct aatcttttat ttttattttt 8400 taggtggagt ctctctctgt cacccaggct ggagtgcagt ggcacgatct cggcttactg 8460 caacctccgc ctcccaggtt caagcaattc ttctgtctca gcctcctgag tagcggggac 8520 tacacgagtg caccaccacg cccagctaat ttttgtattt ttagtagcga cgaggtttca 8580 ccatattggc caggctggtc ttgaactcct gacctcgtga tccgcccacc tcagtctccc 8640 agagtgctgg gattacaggc atgagccact gcgcctggcc tgtttgtttt ttttgttttt 8700 tgtttttttt tgagatggag tctccctctg tcaaccaggc tggagtgcag tggtgcaatt 8760 tcggctcact gcaacctccg cctcctgggt tcaagcgatt ctcctgcctc agcctccaga 8820 gtagctggga ttacaggctc ctgccaccat gcccggttat aatttttaat ttttttttgt 8880 ggaaaaaaag tctccctatg ttgctcaagc tgatcttgaa cttctggctt gaagtgatta 8940 tcctgtcttg acctcccaaa gtgttgggat tacagttgtg agccgttctc ttgtatgaac 9000 acttgtcact gaatttaggg cccaggtgga taattcagaa taatctcatc ttgacatctc 9060 tcatttaatt acatctgcat agccaggcct ggtggcgtgt gcctgtactc ccagctacgc 9120 aggaggctga gacaaagaat tgcttgaacc ccgggaggtg gaggttgcag tgagccaaga 9180 tcgcgccact gcactgcagc ctgggtgaca gagcaagact ccgtctctta aaaaaaaaaa 9240 attacatctg caaagagcca tttaaaaaaa aagtaaggtc ctagtcacag gttctgggac 9300 atggatgtca tcttttcagg ggctgtgacc caactgacta catccttcat ggttctgatg 9360 gctttcaccg ccccctgcca gcatgttgta tattaatttg ttattcgtat atgttctcct 9420 tgcctggacc tggctgcaag gatccagagg gatagggacc cctttcattg tgtttgttgc 9480 tgtatttgta ttgctcagaa cgtagtaggt actcagtaaa tattcgttgc atgaatgaat 9540 gtgtgttttg ttttgttttg tttctttgag accgagtctt actctgtcac ccaggctgca 9600 gtgcagtggc gcgatcttgg ctcactgcaa cctccacctc ccgagttcaa gcaattctcc 9660 tgcctcagcc tcttgagtag ctgggactac aggcacgcac cactatgccc agcaaagttt 9720 cgtactttta gtagagacgg ggttacacca tgttggctag gctggtcttg aactcctgac 9780 cccaagtgat ctgcccgccg cagcctccca aagttctggg attacaggcg tgagccactg 9840 ctcccagcaa atgtgtgttt gctgctctgt ttccctgcgt gtttcttgcc tgtcttgtgt 9900 gtatctttgt gtctggggcc atcctgccct gtgctgccca accaagccat ctctgcccac 9960 aggtgcccgc ctggctgggt gggtgagcgg tgtcagctgg aggacccctg tcactcaggc 10020 ccctgtgctg gccgtggtgt ctgccagagt tcagtggtgg ctggcaccgc ccgattctca 10080 tgccggtgcc cccgtggctt ccgaggtgag aggggaagag tctggagggg aggtagtcgg 10140 gggtgtggtc agtcctaaac tcaccctgtc ctggtccctc caggccctga ctgctccctg 10200 ccagatccct gcctcagcag cccttgtgcc cacggtgccc gctgctcagt ggggcccgat 10260 ggacgcttcc tctgctcctg cccacctggc taccagggcc gcagctgccg aagcgacgtg 10320 gatgagtgcc gggtgggtga gccctgccgc catggtggca cctgcctcaa cacacctggc 10380 tccttccgct gccagtgtcc agctggctac acagggccac tatgtgagaa ccccgcggtg 10440 ccctgtgcac cctcaccatg ccgtaacggg ggcacctgca ggcagagtgg cgacctcact 10500 tacgactgtg cctgtcttcc tggtgagtga gccctactca ggagagtcag aggggtgggc 10560 gtggggacag caggccagcc cggcggtgac catccttgcc cccttccctg ctagggtttg 10620 agggtcagaa ttgtgaagtg aacgtggacg actgtccagg acaccgatgt ctcaatgggg 10680 ggacatgcgt ggatggcgtc aacacctata actgccagtg ccctcctgag tggacaggtg 10740 ggcactgcgg ccagagggag cggggaggca ggcctcgggt ggacatgcgc caggtggctg 10800 gactgctgca tctgtgtgcc acaggccagt tctgcacgga ggacgtggat gagtgtcagc 10860 tgcagcccaa cgcctgccac aatgggggta cctgcttcaa cacgctgggt ggccacagct 10920 gcgtgtgtgt caatggctgg acaggcgaga gctgcagtca gaatatcgat gactgtgcca 10980 cagccgtgtg cttccatggg gccacctgcc atgaccgcgt ggcttctttc tactgtgcct 11040 gccccatggg caagactggt gagtggccgt tttctctgca gggagccatg gatggttttt 11100 agtgagggca aataagaagt ctgacttgag tgttagaaag attatgctag gctgggcaca 11160 gtggctcatg cctgtaatca tagcactttg ggaggcccag gcgggcggat cactggggtc 11220 aggagtttga gaccagcctg gtcaatatgg tgaaacccca tctctactaa aaacacaaaa 11280 attagctgcg cgtggtggtg cacacctgta atccaagtct ctcaggaggc taaggcacga 11340 gagtagcttg aacccaggag gtggagattg cagtgagcca agattgcacc actgcactct 11400 agcctgggtg acagagtgag actccttctc aaatttaaaa aataaaaaaa agattatgct 11460 aggccgggca cagtggctca tgcctgtaat cccagcactt tgggaggcca agatgggagg 11520 atcgcttggg cccaggactt tgagaccagc ctggacaaca tagtgagttt ttgtcatctc 11580 tacaaaaact tggtaggctg ggtgtggtgg ctcacgcctg taaccccaac acattgggag 11640 gcccaggcgg gtagattgct tgagctcagc agtttgagac cagcctgggc aacatggcaa 11700 aaccctatct ctaccaaaaa tacaaaaaat tagctgggca tggtagcgca tgcctgtagt 11760 cccagctact ctggatgcta aggttgaagg atggcttgag cccagaaggt ggaggttgca 11820 gtgagctgag actgagccac tgcgttccag cctgggagac agtgtaagac cgtgtataac 11880 aaaaaagaga gagaaaaaaa gatgatgctg gaggcagctt tagtggggaa gtgtaagcct 11940 gggagaggct tgggaggagg ctgacattgc actgatgcat ggctcagggc agaagccatt 12000 ggaatgggga actcctggga gttacctttg gaagtggcaa agatcagaga acatgtttag 12060 gaggggaacg tggatataga aagagggcat acatggcaga gggtattaca gaagcaaagg 12120 ctggagtgta ggaccctttt gtaatcagaa gagtcaaaag gttttaaaac aatatgtgga 12180 tgtttgcaat ttattttatt ttatttttga gacagagtct cactttgtca cccaggctgg 12240 agtacagtgg catgatctca gctcactgca gcctcgactt cctgggctca agtgatcctc 12300 ccacctcagt cccccaagta gctgggatta caggcgtgcg ccaccatgcc cggctaagct 12360 ttgtattttt tgtagagacg ggtctcacta tattgcccag gctggtcttg aactcttgaa 12420 ctcaagcgat gcactcacct tggcctccca aagtgctagg attatagacg tgagccactg 12480 tgcccggtct gcaatttatt tttaaatggt ttagaaaaga agattggtag attaagctaa 12540 caatagttga atctaggtgg tgggtatatg aatggtcaac ttttctgtat gtttgatagt 12600 tctcataata aatggattaa aaaagcagct actaaatact gtttaggttt aataaaaaca 12660 acaggccggg cgcggtggct cacgcctgta atcccagcac tttgggaggc tgaggcaggc 12720 agatcacctg aggtcaggag tttgagacca acctggctaa cttggtgaaa ccccgtttct 12780 actaaaaata caaaaaatta gccgggcgtg gtggcacatg cctgtaatcc cagctactcg 12840 ggaggctgag caggagaatc gcttgaacct gggaggtgga ggttacagtg agccaagatc 12900 gcgctattgt actccagctt gggcaacaag agcgaaactt cgtctcaaaa aacaaaacaa 12960 aacaacaaca acgacaaaac aatactcaag gggtgtgggc cttttgggca gagcaggaag 13020 atctgcctat gacttctgct taccacttcc caggcctcct gtgtcacctg gatgacgcct 13080 gtgtcagcaa cccctgccac gaggatgcta tctgtgacac aaatccggtg aacggccggg 13140 ccatttgcac ctgtcctccc ggcttcacgg gtggggcatg tgaccaggat gtggacgagt 13200 gctctatcgg tgaggggagc tccatcgtct gtgaatgggc tgggaaagag gggagaggag 13260 ggggtcccgg ccagccacgc ccacaccgat cgcactccat ccggcaggcg ccaacccctg 13320 cgagcacttg ggcaggtgcg tgaacacgca gggctccttc ctgtgccagt gcggtcgtgg 13380 ctacactgga cctcgctgtg agaccgatgt caacgagtgt ctgtcggggc cctgccgaaa 13440 ccaggccacg tgcctcgacc gcataggcca gttcacctgt atctgtatgg caggtgggtg 13500 gtgggcgtgg cctgggcggg tcctgaggca gggggcgggg acagagaagc ccgcggatgg 13560 ggagctgagc agaatggggt gtaagtgggt ttggagtggg acccttagag actgaagcct 13620 ggaaggaggt ggggtcagga ccagaaggga atactgtgtg attctggggg cttggtcaaa 13680 tggagtcagt agaacctcag gtgggctggg agtattcttg gccttaggac ccactcggga 13740 aggggttggg gatagagagg cagagtcggg gctgctgtga ggtcaaggtg aagcctgcag 13800 acaagcttgc agtaagcctg ggcaaaggct atggggggcc ctggggctgg gggctgggga 13860 ctggggaaag ggcttagtct gaggactggg gagaggagga ggggtttgaa ggcaaggcct 13920 ctgcggacac ccatgagcct tgggccagtt gggaccacgg ctgggtgaca gtccagcctg 13980 tggctgaaaa ttaaggttgg gctgggagtg tagaggtggg gacgaaggct cgggggattt 14040 gtcgatgagt aggaagggaa gtacctcctt ccttgcaccc cgttcacacc atagggtagc 14100 ccccgctttc ctaagcccta ttccctgccc caggcttcac aggaacctat tgcgaggtgg 14160 acattgacga gtgtcagagt agcccctgtg tcaacggtgg ggtctgcaag gaccgagtca 14220 atggcttcag ctgcacctgc ccctcgggtg aggacctcag gagagggagc ccgaaaagac 14280 atgtctggga aggggcaaaa actccagggt gggaacctgt aaaaccacgt tagcggataa 14340 cttacaatag aaaccatatg ttatgaaaca ttgaagttaa tttaaatcca agtaaatggg 14400 tttttccacc caaacaaggc ggggctggag aggggtgtac tgctctcacc ctttctgggc 14460 ctctgctcat cccactcccc accccaggct tcagcggctc cacgtgtcag ctggacgtgg 14520 acgaatgcgc cagcacgccc tgcaggaatg gcgccaaatg cgtggaccag cccgatggct 14580 acgagtgccg ctgtgccgag ggtgaggcgg gccaatgaca gtccgacaag aatcaggagg 14640 cggggcttgt gggcgacagg gccaataaca gacttgggga gagcttgggg gcggggttgt 14700 agcaaggcag gacccagtga cagagttggg gggtggagcc aataatgcat atagactgac 14760 ataaggctac gtgtggagcc aaagtgttgg gctgggcaca gggtgcctgt aactgaatgg 14820 ggagggccca ataccttccc ctggcacctg ccatgtgctc ccatctccag tggagggtgg 14880 ggcagaaaca gggctggagg tggggccaca gctgggggcg ggatttaact gtgggagagt 14940 cttggctagg gacaaattct ggagcttgta gtggggtgga gtggaagtaa gtggggggtg 15000 gggggtgggg cctgtattga gctcgcctcc tgacagcttg atgggcaggg cctcagatag 15060 agctgaacca ggattggtcc gaggcctcac ttgtgggcag gcccctggca agtgggcgga 15120 gcctgaccct cttggcccca caggctttga gggcacgctg tgtgatcgca acgtggacga 15180 ctgctcccct gacccatgcc accatggtcg ctgcgtggat ggcatcgcca gcttctcatg 15240 tgcctgtgct cctggctaca cgggcacacg ctgcgagagc caggtggacg aatgccgcag 15300 ccagccctgc cgccatggcg gcaaatgcct agacctggtg gacaagtacc tctgccgctg 15360 cccttctggg accacaggtg ggaccggggg ctggggcaga aacagcacac ctggaggggc 15420 acagagggtt tgggaatggt gcatctagtg gggcacagtg gtggccactc catgccatgt 15480 tcctggcccc taggtgtgaa ctgcgaagtg aacattgacg actgtgccag caacccctgc 15540 acctttggag tctgccgtga tggcatcaac cgctacgact gtgtctgcca acctggcttc 15600 acaggtgggc aagtggctgc catgagaggg ggtccttaga tcgagggtga agtcactcgt 15660 ctctgtgggc ctgttttgcc cgtatctttg cagactcttg tccaacgagg ttgtccgtgc 15720 tttgccctga gtctgtgctg tctcattggc ataaggttgt ttcgagatta ttctgcaggc 15780 tgggcgcggt ggctcacgcc tgtaatccca gcgctctggg aggccaaggc aagtggatca 15840 cttgaggtca ggagttcgag accagcgtgg ccaacgttgc gaagccccat ctctactaaa 15900 aatacaaaaa ttagtcgggt gtggtggtgg gcgcctgtag tcctagctac tcgggaggct 15960 gaggcaggag aattgcttga cctcaggagg cggaggcggc agtgagccaa gattgtgcca 16020 ctgtactcca gtctgggcga cagagtgaga cttcatttca aacacacaca cacacacaca 16080 cacacacaca cacacacaca catacaaaaa caaacaaaaa gattattctg gaacatagat 16140 gaaagtgtcc aaactcagtg actaggttat ttctgaatgg gtctaaacta atccttagtg 16200 gaaatgaatg tgactttgct aggtgtgttg gctcatgtct gtaatccccc agcactttgg 16260 gaggctgaga catgaggatc acttgagccc aggagttcga gactagcctg ggcaacataa 16320 tgaaaccctg tctctacaaa aaataaacac actcccaaag ccagattagt caatacgtag 16380 ataaacacgc acacacatgc acacacaatt agccagacgt ggtggtgcgc aactgtagtc 16440 ccagccactc gggaggctga ggtgggagaa tcgcttgagc ccaggaagtg gaggctgtgg 16500 tgagctatga tcatgccact gcagtccagc ccgggcgaca gagtgagacc ccatctcaaa 16560 aaataagaag aagaagaaaa gaaaaagaaa gggacttcat ggaagtttgg ggaccagaat 16620 gatctggggc aagtcagctc ttggttgagg aggcgggtgt cctaatctgc acaagagctg 16680 atgcgttatg aaaaagaggt cattgctcgg gggtgtgggt gtgctaagtg gggtcacgtc 16740 gtccctccct ggttgtccct gctgactttg ttctgagatg agattgcttg tgtactcccc 16800 agggcccctt tgtaacgtgg agatcaatga gtgtgcttcc agcccatgcg gcgagggagg 16860 ttcctgtgtg gatggggaaa atggcttccg ctgcctctgc ccgcctggct ccttgccccc 16920 actctgcctc cccccgagcc atccctgtgc ccatgagccc tgcagtcacg gcatctgcta 16980 tgatgcacct ggcgggtgag ggcccttctc agcctcagac actgccccct ctccctggcc 17040 cacctccctg gcctgactac cttcccctgc caggttccgc tgtgtgtgtg agcctggctg 17100 gagtggcccc cgctgcagcc agagcctggc ccgagacgcc tgtgagtccc agccgtgcag 17160 ggccggtggg acatgcagca gcgatggaat gggtttccac tgcacctgcc cgcctggtgt 17220 ccagggtgtg tacctcacct tccctctgca gccccaccaa caccatgggc cttctcatct 17280 ctcttctcct ctactctctc ctcccgctat gttagcttct tttttgcttt caatcatttc 17340 cctccaggag cttgggaggt gggtatcaag gtggggaccc tggggttggg ggaggtgggg 17400 ggagatggga tcagggagtc cctcaaggct atctctgctt ccctctcttc cacccccaac 17460 aggacgtcag tgtgaactcc tctccccctg caccccgaac ccctgtgagc atgggggccg 17520 ctgcgagtct gcccctggcc agctgcctgt ctgctcctgc ccccagggct ggcaaggtat 17580 gccacctgct tctcttccct cctcctctcc atctcctctg ccccctaccc tatcagggat 17640 gatgctgggg atccagagag ggacccatcc ccagccctgc ctttgagagg ggcttccttc 17700 ttggaaggac agagctggac actgatatgc agcctgtgtg ggtcagccct tggaagagga 17760 agagttatgg ggctagagat gctgggactt agggcggggt ttagggtaca gcttcctgga 17820 ggagcagacc tcatagtagg tatttgccac ctactgagac agggagacgg ccattctgaa 17880 actgcatatg caaaatgccc agacacgaat gacagcacgg cttattttgc cttcacccat 17940 ctcggcctgc aggttcttcc tgagccccag gtccctgaga ccctgctctg taccctgtaa 18000 ccctaggtgt aaccttgctc cctaccccca ggcccacgat gccagcagga tgtggacgag 18060 tgtgctggcc ccgcaccctg tggccctcat ggtatctgca ccaacctggc agggagtttc 18120 agctgcacct gccatggagg gtacactggc ccttcctgcg atcaggacat caatgactgt 18180 gaccccagtg agtgcagggg agctcttggg ggtgctgctc tggggaacac agtcattaag 18240 cttgaactgt gtgcctggca ctgtgctgtc atttcatctt cacagttatc ttacttttcc 18300 cgttttattt atatggaaac tgaggcccga ggaggttgag tgacttcttg tgcaagccca 18360 tggaacgagt tagggaatga gctgggctta gaaccttgga gtctgtgtgc tgcctctttt 18420 tttttttttt tttttttttt ttttgagaca gagtcttagc tctgtcgctg gagtgcagtg 18480 ttgcaatcat cgctcactgc aacctctgcc ttctgggctc aagcacctca gcctccccag 18540 tggctgcgac tataggcgca caccacagcg cccagctaat attttgtgtt ttttgtagag 18600 acggagtttc gccatgttgc ccacggtggt ctccaattcc tgggctcaag caatctacct 18660 gtctccacct cccaaagtgc tgggattaca ggcgtgagca accacacccg acctaagtgc 18720 actgtctttg agagcaatcg agttaatatc atgtgcaaag tatgagataa aaggcccccc 18780 agaggccggg cgaggtggct cacacctgta atctcagcac tttgggaggc caaggtgggc 18840 ggatcacctg aggtcaggag tttgagacca gcctggccaa catggagaaa ccccatctct 18900 actaaaaata caaaattagc agggcatggt ggcgcatgcc tgtaatccca gctacttggg 18960 aggctgaggc aggagaatca cttgaacctt ggaggtggag attacggtga gctgagattg 19020 tgtcattgta ctgcagcctg ggcaacaagg cataactccg tctcaaaaac gaacaaacaa 19080 acaaacaaac aaacaaaaag gggtgctgcc aaacagataa ataacacaag ccacatatgc 19140 aattttaaat tttctagtag ccacattaaa aaataaacag aaatgggaga tattaaattt 19200 aatattttat atttatttat ttattatgta ttcagagtga ggctctgtcg cccaggctgg 19260 agtgcagtgg tgcgatcttg gctcagtgca gtctcgaact cctgggctga ggcattcctc 19320 ctgtcttaac ctcctgagta gctaggatta caggcactca ctacatgcct agctaatatt 19380 tttattttaa ttctgtagag atggggtctc actgcgttgt ctaggctggt ctcaaactcc 19440 tggcctcaag ccatcctcct gccttggact ctcagactgc tgccactgtg cctgaccaaa 19500 tttaatatat ttaaatttaa tttattgaat gtatataatc ggctgggcgc agtggctcac 19560 accggtaatc ccagcacttt gggaggctga ggcaggtgga tcacctgagg tcagaagttt 19620 gagaccagcc tgaccaacat ggagaagccc cgtctctact aaaaacacaa aattagctgg 19680 gcgtggtggc acatgcctgt aatctcagct actcaggagg ctgaggtagg agaatcactt 19740 gaacccggga ggcggaggtt gtggtgagct gagatcgtgc cattgcactc cagcctgggc 19800 aacaagagcg aaaccccatc tcaaaaaaaa taaaaataaa aataaaagta aataaataaa 19860 taaataaata aatgtatata atctatttaa cccaatatat taacatatca tttcaacatg 19920 taaccagtat aaaattgtta atgagatatt ttacattgtt tccttttttt tttttgagac 19980 aagttctcac tctgtcgccc agactggagt gcagtggtgc gatcatgact cactgcagcc 20040 ttgacctccc aggctcaagt gatcctcccg tctcagcctc cccactagct gggactacag 20100 gtgtgcacca tcatgcccag ctaatttttg tattttttat agaaatgggg tcttgccatg 20160 ttgcccaggt tggtctcgaa ctcctgagct caagtgatct gcccacctcg gcctcccaaa 20220 gtgctgggat tacaagtgtg tgccaccacg cccagcctat tttttttttc ataccaagtc 20280 ttcaaaatct gaaacccagt atgtatttta catttatagc acatctccat ttagtcacaa 20340 tttcaaggac tcagtagcca catgtggcta atggccactg tccttgtcct gttccaagca 20400 caggaattag atcaggcaca attagcagcg tttgcttggc agcttaaagg gaccttttgg 20460 ttttcccaac atcctgccct tgccacatag gtgaggtttc cagataaagg aggggacgag 20520 gccacagaag gggatggatt tagattcctc tgaccaaatg caccccatcc cagttgaacc 20580 tggtttcctc ctgaattctt ctcaatccaa ggcatatccc agtcagactg ggctaatggg 20640 ggcaaggtag gtgaccagac cgccttcctc ctgtccgcag acccatgcct gaacggtggc 20700 tcgtgccaag acggcgtggg ctccttttcc tgctcctgcc tccctggttt cgccggccca 20760 cgatgcgccc gcgatgtgga tgagtgcctg agcaacccct gcggcccggg cacctgtacc 20820 gaccacgtgg cctccttcac ctgcacctgc ccgccaggct acggaggctt ccactgcgaa 20880 caggacctgc ccgactgcag ccccaggtgg gcggggcctc tgcttggaga gcagggactc 20940 tggcttggga tggggcctgg gacctgggac gaagtgggga cagcaacgac ttgggggagt 21000 cctcagcttg gtacccactg cggactctga tgactgatgg ggcagggcca gggaaggagc 21060 agggcgtctg tttggagctc accttggagt ctctggtgcc tgagagggca tggcttccag 21120 gtggctctca ccttagggct gaagtccctg gtgaattgga gctaagggac ctgattggct 21180 tctgctgggg ctgcagcttc ttgccgagat aagggtcagg gagtgggacg tccccagcgc 21240 taacagcggg actcaggaag gagggcaggg cctgtgaggg ggcggagcct gatcctccct 21300 cccactcctt ccgctccagc tcctgcttca atggcgggac ctgtgtggac ggcgtgaact 21360 cgttcagctg cctgtgccgt cccggctaca caggagccca ctgccaacat gaggcagacc 21420 cctgcctctc gcggccctgc ctacacgggg gcgtctgcag cgccgcccac cctggcttcc 21480 gctgcacctg cctcgagagc ttcacgggcc cgcagtgcca ggtgggtgga gttactgggg 21540 acctggggga ggagcctgcc tgggatccta ggagggagaa gccaagtcgg ggcacagttt 21600 ctcccagact accccccacc ccacagtact gactctgagt gcttcccctc cagacgctgg 21660 tggattggtg cagccgccag ccttgtcaaa acgggggtcg ctgcgtccag actggggcct 21720 attgcctttg tccccctgga tggagcggac gcctctgtga catccgaagc ttgccctgca 21780 gggaggccgc agcccagatc ggtgagtggg agcatgtggg cgggcgtgtg gggccttggg 21840 aaggggctca tgcacgtacc tcctgctagt gtgagccgaa tgggggtgcc acagagccta 21900 tggggcatga tggggtgtgt ggctgagtgg tgtgggtgtg tgagcatgtg acaactggcc 21960 gggatggtgt gtgtctgtca ctgtgagaca gcagggatgg tgtgtgtgct acagggtgtg 22020 accgagctgc tgtgagcgtt gaaggcatgt gtgtgtatgt cagtgatgag accttgcctg 22080 ctggggggat gtgtgacaac ctgatggagt gtgggtgcga tcctggggac tcattccacc 22140 aaggatgttg aatgatctgt gtgatggagg cagaaggggg atgtatgggg ttacctctgt 22200 tcctgtgcca ctctcctctt tgcaggggtg cggctggagc agctgtgtca ggcgggtggg 22260 cagtgtgtgg atgaagacag ctcccactac tgcgtgtgcc cagagggccg tactggtagc 22320 cactgtgagc aggaggtgga cccctgcttg gcccagccct gccagcatgg ggggacctgc 22380 cgtggctata tggggggcta catgtgtgag gtaagggggc gctccccagg agagggaaga 22440 ggaggtgggc atgcttgggt gcgtctgggc acacatttct gtgtggcttg gtatgggtat 22500 gtctctagat atgtctgagt ttttgctttt gtgactcttg gtgtatctgt gggtgtcact 22560 ctgggtagat ctggggtgtt tttttatttg tttgtttttt tgagacaggt tctcactctg 22620 tcacccagac gagtgcagtg gcgcgatctc agctcactgc aacctttgcc tcctgggttc 22680 aggtgattct tctgcttcag cctctcatta tagctgggag tacaggcacc caccaccacg 22740 cccagctaat ttttgtattt ttcatagtga cagggtttca ccatgttggc caggctggtc 22800 tcgaactcct gatctcaggt gatctgccca cctcggcctc ccaaagtgct gggattacct 22860 actggtagtg cgctgaacat ctgtgtgtgt ccttttgtgc tggggttctt tgcgtcttca 22920 tgggtatgtc caggtgggtc tgtgtcccac taagctgagt gggtccctct cttaccccac 22980 tgaagtgtct tcctggctac aatggtgata actgtgagga cgacgtggac gagtgtgcct 23040 cccagccctg ccagcacggg ggttcatgca ttgacctcgt ggcccgctat ctctgctcct 23100 gtcccccagg aacgctgggt atgccagggc cagggttggg gggacaggat gagaggctgt 23160 cttcattccc tcttgaccac ccctcgtttc ttcccccagg ggtgctctgc gagattaatg 23220 aggatgactg cggcccaggc ccaccgctgg actcagggcc ccggtgccta cacaatggca 23280 cctgcgtgga cctggtgggt ggtttccgct gcacctgtcc cccaggatac actggtttgc 23340 gctgcgaggc agacatcaat gagtgtcgct caggtgcctg ccacgcggca cacacccggg 23400 actgcctgca ggacccaggc ggaggtttcc gttgcctttg tcatgctggc ttctcaggta 23460 agcgttggcg aaggggctgg cctgggaccc cgcctgtcat tcccccattg tggctgatct 23520 acatgctccc gctcgctcag gtcctcgctg tcagactgtc ctgtctccct gcgagtccca 23580 gccatgccag catggaggcc agtgccgtcc tagcccgggt cctgggggtg ggctgacctt 23640 cacctgtcac tgtgcccagg taggtgtggg tggcggcctt tggaggagga gtaggggcgt 23700 ggcctctgga gtagtagggg cgtggcgtct aggaggagga tgtggcttta ggggagacat 23760 cgaagggaag ggagtttctg gaaaatgctg acatttccgc cgggtgtggt agctcacacc 23820 tgtaatccca gcacattggg aggccgaggc gggaggatca cttgaggcca ggagttagag 23880 accagcctgg gcaacatggt gaaaccccgt ctctactaaa aatataaaaa ttagccgggc 23940 gtagtggcag ctgcctgtaa tcccagctac tcgggaggct gaggcaggag aatcacttga 24000 acccgggagg cggaggttgc agtgagccat cacgccattg cactccagcc tggcgactga 24060 gtgacactcc gtctcaaaaa acaaagaaac aaccccctgc cccgacattt cctggaggtt 24120 tgaagggaaa agggtgagga tggtgattgg ggggcgtggc ctcctgggat ggcagggctt 24180 atctgccagg tggggtctcc agtgtggaaa ggggagcggt tgggtgggac atggggaggt 24240 tgagggggtc tcaaccttcc ttagtcttga cctcttctct tccccctctc tccccttgac 24300 tcttcttttc cccactcctc catttcttct cctccttccc tccactcccc accctcattt 24360 ttatccctcc ctccccaaac ccgaccccca gccgttctgg ggtccgcgtt gcgagcgggt 24420 ggcgcgctcc tgccgggagc tgcagtgccc ggtgggcgtc ccatgccagc agacgccccg 24480 cgggccgcgc tgcgcctgcc ccccagggtt gtcgggaccc tcctgccgca gcttcccggg 24540 gtcgccgccg ggggccagca acgccagctg cgcggccgcc ccctgtctcc acgggggctc 24600 ctgccgcccc gcgccgctcg cgcccttctt ccgctgcgct tgcgcgcagg gctggaccgg 24660 gccgcgctgc gaggcgcccg ccgcggcacc cgaggtctcg gaggagccgc ggtgcccgcg 24720 cgccgcctgc caggccaagc gcggggacca gcgctgcgac cgcgagtgca acagcccagg 24780 ctgcggctgg gacggcggcg actgctcgct gagcgtgggc gacccctggc ggcaatgcga 24840 ggcgctgcag tgctggcgcc tcttcaacaa cagccgctgc gaccccgcct gcagctcgcc 24900 cgcctgcctc tacgacaact tcgactgcca cgccggtggc cgcgagcgca cttgcaagtg 24960 agcccatcca cccgatcgat ccgtctgtct atgcatccat cccagtctgt ttgtccgtgg 25020 gccctgcctc tctttccacc tgcccatcca cttgccccgt ctgtttctct gtgcactcta 25080 tctgcccatc ggtgtgtccc acgtgtctgt ccgtgtgtct gttcatccat gtgctctgtc 25140 tatcttgttc ttgtttctat ctgcctatgc acttctctgc cccgtttgtc tgcccttttc 25200 tccatccaat aattactttt tttttttttt cccgagatgg agtcttgctc tgtggcccag 25260 gctagagtgc agtggcgcga tctcggctca ctgcaacctc tgcctcccgg gtttaagcag 25320 tactcctgcc tcagcctctg gagtagctgg gattacaggt gtgagccacc gtgcccagcc 25380 gaattccttt tttttttttt tttttttttt aagacggagt ctcgctctct tgcccagact 25440 ggagtgcaat ggctccatct tggctcactg caacctccgc ctcccgggtt caagcgattc 25500 tcctgcctca gccttccgag tagctgggac tacaggtacc tgccaccacg cctggctttt 25560 tttttgtatt tttagtagag acagggtttc accatattgg ccaggctgct cacgaactcc 25620 tgaccttgtg atccgcccgc cttggcctcc caaagtgctg ggattacagg tgtgagccac 25680 cgcacctggc tccttttttt tttttttttt ttttttttta agacagggtc ttgctctgtg 25740 gcccaggctg gagtgcagtg gtgtgatctt ggctcagtgc aatctctacc tcctgggctc 25800 aagtaaccct cacacctcag cctccctagg agctgggacc acaggtgtga gccaccgcgc 25860 ccgggtaact tttgtgcttt ttcttagaga tggggtttcg ccatgttgtc caggctagtc 25920 ttgaacacct gagctcaaag caatccaccc agcttagcct ctcaaagtgc tgggattgtg 25980 ggcctgagcc accacatcta gcctaaaaat tccttttatt ggccgggcac agtgctcacg 26040 cctgtaatcc cagtactttg ggaggcagag gagggcagat cacctgaggt caggagtttg 26100 agcttgggca acatggtgaa actccgtctc tactaaaaat acaaaattag ccgggtgtgg 26160 tggcacatgg ctgtaatccc agctactcag gaggctgagg caggagaatt gcttgaacct 26220 gggaggtgga ggatgaggtg agctgagatc gtgccattgc actccagcct gggcagcaag 26280 agtaaatctc tgtctcacca aaaaaaaaaa aaaaaaaaaa aattgctttt gttcctctgt 26340 gtacccaact gtcttcctcc actcctgttt gtccatcctt gggccgaatc tgtccatcca 26400 tgcactcttt ttgctcctct atatggctat ctgtgggtgc ccctgactat ctctgcccct 26460 cttctgcccc tctctctgtc tgtcctaggc agtccatctg tccatcccta caccctgccc 26520 cttctgtcct ctgcgtacct ctctgtctgc ttggaaccct ccaccccctt aatctccaca 26580 tcctcatctt ctctgctttc ccctcactca ctctgttcca gccaccctgg cctctctgct 26640 gtttcccaga taccccaggc ttgatcccat atcagggcct ttgcacaagc tgttcctact 26700 gcctggctgt tcccttcccc cacatggctc ctccctcacc tccttcaagt ctttgctgaa 26760 atggtggtac cttctcagtg agcacctccc tggtcacccc cagcccttcc tctgctttat 26820 ttttctgctt agcatttaat tccatccagc actatgtgtg tgcgtgcgtg tgtgtgtgtg 26880 tgtgtgtgtg tatatacata tatatacata tatatatata tatatgtatt tttttttttt 26940 ttttttttag acaggatttc tctcccattg cccaggctgg agtgcagtgg tgtgatcttg 27000 gctcactgca acctccacct cctgggttca agctattctc ctgcctcagc ttcccgagta 27060 gctgggatta caggcgcccg ccaccatgcc tgactaattt ttgtattttt agtagagacg 27120 ggggtttcac cattttggcc aggctggcct agaactcctg acctcaggtt atccgtacac 27180 cttggcctcc caaagtgctg gaattagagg catgagccac cgcacccagc ctgtattttt 27240 attttattga tgagtaatgg agatgctgta tatttggctc atgtatctcc cttactatct 27300 gtcttcaccc agtggaatgc cagctgcacg tggaccagga attgtggccg ggccttatcc 27360 ttgtgcctgc agcagtgccc aacacatggt agtcagttag tatttactga ttaattaata 27420 gttagccagt gaggccggga gtgtggtgtg ggtttgtagt cctagctact tgggaggctg 27480 aagagggagg ataatttgag tccaggagtt tgagattgca gagagctatg attgcaccac 27540 tgcactccgg cgtgggcaac agagcaagac cccatttcaa tcaatcaatt taaaaaaata 27600 ttatcaaggg ctgcgtgcgg tgacttatgc ctgtgatccc agcactttgg gaggccgagg 27660 caggtggatc acctgaggtc aggagttcaa cagcagcctg gccaacatag taaaaccccg 27720 tctctgctaa aaatacaaaa attagacggg cgtggtggtg catgcctgta atcccagcta 27780 cttgggaggc tgaggcggga gaatcgctgg aaccggggag gtggaggctg cagtgagctg 27840 agagatcatg ctactgcatt ccggcctggg cgacagagca agactccgtc tcaaaaaaaa 27900 aaaaaaaaaa ttatccagtg aatggcaaga tttgcagaaa gcctggcatg ggtccgtata 27960 ttccagtccc ctgtgcaagc cttgtctgga gtctctgtac tctacggtgt gaatgcatgg 28020 aaggggattg tctcctctga cccctgactc cgcccctctc tgcctcaccc ttccccacca 28080 gcccggtgta cgagaagtac tgcgccgacc actttgccga cggccgctgc gaccagggct 28140 gcaacacgga ggagtgcggc tgggatgggc tggattgtgc cagcgaggtg ccggccctgc 28200 tggcccgcgg cgtgctggtg ctcacagtgc tgctgccgcc agaggagcta ctgcgttcca 28260 gcgccgactt tctgcagcgg ctcagcgcca tcctgcgcac ctcgctgcgc ttccgcctgg 28320 acgcgcacgg ccaggccatg gtcttccctt accaccggcc tagtcctggc tccgaacccc 28380 gggcccgtcg ggagctggcc cccgaggtga tcgggtgagt gaccccacct ggaaaagccg 28440 tggtggctgg ggagaggaga gatgctgttg atccaggtct tgtgtgtttc atttcactta 28500 atgcctttac ccaccagatg tcaggagcac cgcctcctgg ctgtgacaag caaaaaatgc 28560 ctccagatgg agctagtgct tctggggtag aattgcccag gtctagtgct ggcttgtggg 28620 gaggcaggtt gctaagtggc ttggcagagt ggttaaatac atgtgtaggg tttttttctt 28680 cttcttcttc ttcttctcct tctccttctc cttctccttc tccttctcct tcttcttttc 28740 tttttttttt tttttttgag acggagtctc tgtcgcccag gctggagtgc agtggcgtga 28800 tctcagctca ctgcaacctc tgcctcctgg gttcaagcaa ttctctgcct cagcctccag 28860 agtagctggg attacaggca tccaccacca caccgggcta attttcttct tcttaaagac 28920 atagaagata aagccgggca caatatattc ttgtagtccc agctacttgg aggatcactt 28980 gagcccagga attcaaatct agcctgggca acatagcaag actcccatct ctctctctct 29040 tttttttttt tttttttttt gagacggagt ctcgctctgt cgcccaggct agagtgcaga 29100 ggcgcgatct cggctttctg caagctccgc ctcccgggtt catgccattc tcctgcctca 29160 gcctcccgag tagctgggac tacaggcggc cgccaccatg cctggctaat tttttgtatt 29220 tttagtaaga gacggggttt cagtgtgtta gccaggatgg tctggatctc ctgacttcgt 29280 gatccacctg cctcggcctc ccaaagtgct gggattacag gcgtgagcca ccatgcccgg 29340 cctccgatct cttaaaaaaa aaaaaaaaaa aaaaaaagaa gaagaagaag aagaagaaga 29400 agaagaagaa gaagaagaag aagaagaggg aggagggagg gggaggggga gggggaggaa 29460 gaggaagaag aaggaagagg gggaggggga ggaggaggag gaagaggaag aagaagaaag 29520 aaataggagg ggctcggtgg ctcacgcctg taatcccagc actttggaag gcagttcatt 29580 tgacgtcagg agtttgagac cagcctgggc aacatggcaa aaccctgtct ctactaaaaa 29640 tacaaaaaat tagccaggcg tggtggtgta tgcctttagt cccagctact caggaggctg 29700 aggcatgaga atcacttgaa ctcaggaagt gaaggttgca gtgagccaag attgtgctac 29760 tgcactccag ccggggcaac agagccagac tctgtctcaa aaaaaaaaaa aaagaaaaag 29820 aaaaaatata gaagtatcct gctagtggcc tggtagaggt accagaaggg aggtgtagct 29880 attgttgagg agaaggcaga gacctgagag gggtgggtct ctgatacaga gaggagggtt 29940 gggttttgca gatattggga tgtgggtggt ggaaggggag gggtagaggg atgaaggaga 30000 gtggttggtg taccagcccc tggctaagct gagtgatagg gccacaaagt gaaaaacagg 30060 tcattttgga gcttgcagag tctgagggac ctggaagtat tgggaaggcc tccaaggaga 30120 tacctggggg ctaaagacac aaacaagaaa gatataaaag tgttgatgat cattgaagcc 30180 atgagaggga ctcaaaggag tggggagaga aggaacagaa gaattaatct ggggacacgg 30240 agacctaagg attggatgaa tggaattagc tcaaaaaaga ggaagaggcc aggtgtgatg 30300 gctcacaact gtgatcccag cactttggga ggttgaggcg ggtggatcac ctgaggtcgg 30360 gagttcaaga ccagcctgac caacatggag aaaccctgtg tatactaaaa atacaaaatt 30420 actggggcgt ggtggcgcat gcctgtaatc ccagctactc aggaggctga ggcaggagaa 30480 ttgcctgaac cctggacgtg gaggttgtgg cgagctgaga tagcaccatt gcactccagc 30540 ctgggcaaca agagtgaaac tctgtctcaa aacaagcaaa caaacaaaca aatacaatgt 30600 cctggccaac acggtgaaac cccgtctcta ctaaaaatac aaaaattacc tgggcgtggc 30660 agcgcgtgcc tgtaatccca gctactcagg aggctgaggc agagaatcac ttgaaccagg 30720 gagtcggagg ttgcagtgag ctgagattgc accactgcac tccagcctgt cgacagagca 30780 agacttcatc tcaaaaacaa aaaacaaaca atgtcccttg gctgggcgta gtggcccagg 30840 cctgtaaacc cagcacattg ggaggctgag gtgggaggat tgcttgagcc ccggagttca 30900 agactagcct gggcaacata gtgacaccta atctctatgc ctccaccccc aacccccccc 30960 aaaaattagc caggtgtggt ggcacatgtc tgtggtccca gatctttggg agaatgaggt 31020 ggaaagattg cttgaaggcg ggaggtcaag gctacagtga gctctgattt cgccactgtg 31080 ctctagcctg ggcgacagag tgaaaccgtc tcaaaaaaca aaacagaaca aacaaacaac 31140 aaaaaaaaca ggccaggcac agtggcggct cacgcctgta atcccagcac tttgggaggc 31200 caaggcaggg aggatcactt gaggtcagga gttcaagacc agcctggcca acgtggtgaa 31260 accccgtctc tactaaaaat acaaaaaaat tagctgggca tggtggcaca cgcctgtaat 31320 cccagctact tgggaggctg aggcaggaga atcgcttgaa cccaggaggg gaagttgcag 31380 tgagccgaga tcgcaccact gcactccagc ctcggtgcaa gatcaaaaaa taatatccat 31440 tggattcatg gctctggggg gactgctgac cacagcaaga cacgttttag gggtgcagtg 31500 gggttggatg ccaggtggat gcaggtgggc catggagacc tgtgggtgga gatggcgctt 31560 cagagccagg cgctggggag ggaaagtggg gcttggtacc agggggtgca tcgggcaatt 31620 tttgagccct ctggtccctc cctgctgtcc ctgcagctcg gtagtaatgc tggagattga 31680 caaccggctc tgcctgcagt cgcctgagaa tgatcactgc ttccccgatg cccagagcgc 31740 cgctgactac ctgggagcgt tgtcagcggt ggagcgcctg gacttcccgt acccactgcg 31800 ggacgtgcgg ggtgcggccc tgccttgggg agggggtggc gggggcggag ctgggggcgg 31860 ccgaagcccc gcctgaggcc aaagccccgc cctcggctga agccccgccc tctgcttcct 31920 gctcttaggg gagccgctgg agcctccaga acccagcgtc ccgctgctgc cactgctagt 31980 ggcgggcgct gtcttgctgc tggtcattct cgtcctgggt gtcatggtgg cccggcgcaa 32040 gcgcgagcac agcaccctct ggttccctga gggcttctca ctgcacaagg acgtggcctc 32100 tggtcacaag ggccggcggg aacccgtggg ccaggacgcg ctgggcatga agtgagaacc 32160 ctgctcgctc cctgtccctg actacgggga ccttgtgaac cctggacccc gccttgacct 32220 gactcagacc tctgacccca ccccaaatcc tccttcccag ctggacacct ctagtgtccc 32280 cctcacatcc cctcttccca ttgtccgcca ggaacatggc caagggtgag agcctgatgg 32340 gggaggtggc cacagactgg atggacacag agtgcccaga ggccaagcgg ctaaaggtac 32400 tgccccccct ctgacctttg ccccctcctc tgacccctcc cctcagggta ctgggtgggg 32460 tccccagtgg atgatgggcg tgatcagggg atggcaccgc tgtccccacc tccctagctc 32520 cagagaatag tcccagcttt gcaacccttt cttctcagtg tggttctgtg acctcagagg 32580 gaggaagatt tgccactggg gccccaaggg tccctccggg caggtggaaa atcctcccct 32640 ccatcctgcc cctccccagc caggatcctg cttcctggcc agcctgcact cttcctggag 32700 gtgtcccccc agcccagaga gcgagtctgc cttatctctg tcagttccta ttttgtccag 32760 catggactta gcctgaaagt gctctgagcc ggttctgagc tcatggagtc atgcccctgg 32820 gttcagtatg agtcagcttt ggctgccatt aaaaaatccc acagaggtgg atcacctgag 32880 gtcaggagtt cgagaccagc ctgaccaaca tggtgaaacc ccgtctgtac taaaaataca 32940 aaagattagc tgggcatggt ggcgggcacc tgtaatccca gctacttggg aggctgaggc 33000 aggagaatcc cttgaacctg gcactccaga ctgggcaaca agagcgaaac tccgtctcaa 33060 aaaaaaaaaa aagtcccaca gcatgggtga cctaaacaac agaaattaat tttttcacgg 33120 ttctggaggt tggaagtcca agcccaaggt gctgtcaggg ctggttcctg gtaagggctc 33180 tcttcctggt gtgcagatgg ccgccttctc actgtagcct cacatggcct ttcctctgtg 33240 tacacagagg ggagagagag agagagagag agagagagag agagagagag agagacagat 33300 ttgacatccc atcctcttct tgtaaggata ccaatcctat tggattaggg tcccacattt 33360 ataacctcat ataaccttaa ttacctcctt aaaggcccta tctcaccagg cttggtagct 33420 tgcaccagta atttcagcta cttgggaggc tgaggcagga ggatcacttg agtccaggag 33480 ttggagcctg cagtgagcta tgactgcatc acttcactcc agcctgggta acagagcaag 33540 accctgtcta aaaataaagt cctatcttca aatgcaggca catggaaatt ttatatatta 33600 tttagggttt tagtgtatga tttctgttta tttatttatt tattttttga gaccaggtct 33660 cactctgttg cccaagctgg agtgcagtgg cgtgatcttg gctcattgca gcctccacct 33720 cccaggttca agtgattccc ctgcctcagc ctcccgagta gctgggatta taggcatgtg 33780 ctaccatgcc cggctaattt ttgtattttt agtagagatg gggtttcacc atgttggcca 33840 ggctggtctc aagctgcaga cctcaagtga tccacccgcc tcggcatccc aaagtgctgg 33900 gattacagac gtgagccacc gtgcccggcc attgtatgat ttttagaggg ggcacaattg 33960 agtccataca gatcctgcta gcattactga ggcacccgct gaatgtctct aatgcacata 34020 aaacatctta tttcatctct ctgaggacgc tgtaaagtag ctattgacat atacttcaat 34080 ttacaaactc ccttcctctg tccatatttt aatgttgtga aatggaatca tctattacag 34140 tggacagata aaaaaacctg ctcaacccag acttttcctt ctgccttccc atcccaaagt 34200 aggtattaga ggtattaaag gggtggccca atgtaattgt ggtggtatct tagcatggtg 34260 gaatggctgc gtcatcctga ttttgccaat gagaagctca gagcttcaaa gtgatttgcc 34320 cagagccttc cagctactaa ggggtggggt tggaacttga acctggatca gatgctatcc 34380 caatctgctt atactgtgtg cttatagtcc cagctacttg ggccgctgag gtgggaggat 34440 catttgagcc caggaagttg aggctgcagt gaggtatgat tgcagcactg cactccagcc 34500 tgagtgacac agtgagactc agtctctaaa aaatatatac ataaataatt aaagccattt 34560 attactcaaa aagaccaaaa aaaaaaaaaa aaagaaacct tgtgtcttcc ttttattacc 34620 tccttgggct atggcacaca ttgatttcct gttaatctca gcactttggg aggctgagag 34680 gggctgatca cctgaggtca ggagttcgag accagcctgg ccaacatggt gaaacccctt 34740 ctctactaaa aatacaaaaa attagctgga caccgtggca tgtgcctgta atcccagcta 34800 ctccagaggc tgaggcagga gaatcacttg aacccgggag gcgggggttg cagtgagctg 34860 agatcatgcc actgcactcc agcctggggg acagagcgag actctgtctc aaaaacaaat 34920 aaaacaccaa acagatggta aatgatttcc cagggctctg tgtgtatctc ctaaagagaa 34980 acctgtagga atgcccagcc ccatccctgg cagtggctct ccccagcacc aaagggtgag 35040 atctgagatc cagggtgcct gcccctccag gtagaggagc caggcatggg ggctgaggag 35100 gctgtggatt gccgtcagtg gactcaacac catctggttg ctgctgacat ccgcgtggca 35160 ccagccatgg cactgacacc accacagggc gacgcagatg ctgatggcat ggatgtcaat 35220 gtgcgtggcc caggttagtg acagtgcccc tcccaaaggg atgcccctca cccatcctac 35280 ctgtgagagg ttatttctga ctctgtgttt tggggagaac tgggggagtc tctaagcttc 35340 tgtgaagggt gtgtgtattc agcaacactc ttggttggaa gtgacaaagg tccaactcag 35400 actagcttaa gtaaaacaag ggatttatca actcatttaa ctaaaagtgg cacatttgga 35460 tccaggactc agttttcccc tctctaccct ttgactccaa tcttacaaaa ccttcttaat 35520 gctccagaaa agtctcagga tgcattctga ttggacagac tagggtcacg tgctcatcct 35580 tgagccaatc agtatgacca gggggctgga atatgcaaat tgaccaagcc tgattcacat 35640 acctgtcttt gagctggtgg gggtcgggtc tgctggtgga gcggggaggt cagtctctac 35700 ccagggcttg agaatggaga accggatgca agtggctccc taagaaaaat gaggacaagt 35760 gcttgtgtac agtgtgtgca gtgtagcgga ctatgttgtt tgatcctctt agatccatct 35820 ttgttatgtt tcagggcatt cctgccttag cttcattttg cttatgctcc aaatggcctg 35880 tacttatggc tctcttttga agtccctctg gctgctgggg cctgaagtgc ctgggatttt 35940 atgtccctgg gggcagctct ttttttcccc ccccctttta aaacactcaa atgaattggc 36000 agaaaggggc agctcctaac cagtgggtgc aggagtatga aggggatggt ttctttgcct 36060 tgagttggat tagaactctg gggtacaaca catgttccag agtccctttg gaggatcaag 36120 gtggaataac cctttgggaa tttactggag gttgcacact tgcttgactc ccagagtccc 36180 ttgtcctgtg ttccctgtgc cctaggagta gttctgtgac aaatcacgtg cccacgaatc 36240 ctcattctgg gtgtgggtgt gcagacagga ggatctgcta attgtcattt ttccatgtgt 36300 cccattagct cctaatgggg tacccttgat aacatttcct ggaaaaggcc ctgtgtttac 36360 cttcctgctg acacactcct gtccctgcag atggcttcac cccgctaatg ctggcttcct 36420 tctgtggggg ggctctggag ccaatgccaa ctgaagagga tgaggcagat gacacatcag 36480 ctagcatcat ctccgacctg atctgccagg gggctcagct tggggcacgg actgaccgta 36540 ctggcgagac tgctttgcac ctggctgccc gttatgcccg tgctgatgca gccaagcggc 36600 tgctggatgc tggggcagac accaatgccc aggaccactc aggccgcact cccctgcaca 36660 cagctgtcac agccgatgcc cagggtgtct tccaggtgag ataggcacac actttggacc 36720 tcagagctgg ggcaggcatt agacttacct gggtttgagc cccagttctg cctttcagac 36780 tcattttttc tcatttggaa aatggggata tatgggaata cagtatctgt caagcagctg 36840 ctccctgaac ctcaccaatt tcaggggttg gggtatgggg gttggggact tcatgggact 36900 taggggtgct cctgattcct ctgttcctgc catgacccct cctgctgcat ccactctctg 36960 tcctagattc tcatccgaaa ccgctctaca gacttggatg cccgcatggc agatggctca 37020 acggcactga tcctggcggc ccgcctggca gtagagggca tggtggaaga gctcatcgcc 37080 agccatgctg atgtcaatgc tgtggatgag cttggtaggt tggcagagga atcaagtcta 37140 agctgggttg gtgtcacctg ggccctgagg gtcatgttgg tgcaaattca tacccatgtt 37200 gagacccaat cactgaagct cacgcacaca taaccaggct tcatgaagcc tgcagggtca 37260 tgcagggtca ccactagtcc ttagggtgcc tcagggattt agaaaaaggt gcctttcccc 37320 tagatacttc atttccacct gctttgttag acggacacac tgtacttcca cctgctggaa 37380 gttattataa taaacgtaca catcaggcca tgtgtggtgg ctcatgcctg tgatcccaac 37440 actttgggag gctgaggcag gaggatgact tgaggccagg agtttgagac cagactgggc 37500 aacatagtgg gttctacaaa agttttttga aagattagcc atgtgcggtg gtgcatacct 37560 gtggccctag ctactccaga aactgaggtg ggagggtcgt ttgagcccag gaggttgagt 37620 ctgtgagccg tgattgtgcc cctgcactcc agcctggggg acagactcca tctcaaaaaa 37680 aaaaaacagt ctccagacgt tgccaaatgc tctgggggct gggggcaggg agtttctcct 37740 agttgagagc cacagttcta gggcagggct ggccaatagg actttctgtg atgatggaat 37800 tattctctgc actgtccagt atagtagcca ctggccacat atagtgactt gaaatgtgac 37860 tgaggcaaat gaagaagtaa atgttttagt tcatttaatt tttttctcct tatgttgccc 37920 tttttttaat tttttttttt gagacagagt ctcactcttg tcacccaggc tggagtgcaa 37980 tggtgtgatc ttggctcact gcaacctcca ctacccaggt tccagtgatt ctcctgtctc 38040 agcctcccaa gtgtctggga ctaaaggtgc ccatcaccat gcctggctat ttttttgtat 38100 ttttagtaga gacggggttt cgcaatgttg gccaggctgg cctcaaactc ctgacctcag 38160 gtgatccacc tgccttggcc tcccaaagtg ctggggttat aggcatgagc cactgagtcc 38220 atcctttttt ttaaaaacaa aaaacaaaaa acaaaaaact gctttattga gatataatta 38280 acatgccata caattcaccc atttaaagtg tacaattcaa tggctcttag tataatcaga 38340 gtcatacaac tattaccaca atcaatttta gaacatttca tcacctgaaa aataaattct 38400 caccacttgg ccatcatctg ccaagcccct catctgtcca gccctgtgca accactgatt 38460 tgctttttgt cttcatggat ttgcctgttc tggacatttc atataaatgt aatcatatga 38520 tacgtggtct tttttgtctg cctttttttc agttagcatt atgttctcaa ggttcaccca 38580 tgttgtagca tagttcagct gaataataat ccattgtgtc gatggaccac tttttttttc 38640 ttttattttt agacataggg tatcactctg tcactcaggc tggagtgcag tggcatgatt 38700 acggctctct gcagcctcaa actcccaggc tcaagtgatc ctcccatccc accctcctga 38760 atagctggta ttacaggtgt gtgccagcat acctgggtaa ttcttaaatt ttttgtggaa 38820 atggggtctc actttgttgc ccaggctgat ctcaaactcc tggccttaag caatactccc 38880 accttggcct cccaaattgt tgagattata ggcgtgagcc actgtgcctg gccaaaagtt 38940 tcaattttga tcatgtccaa tttatctgtt ttgtagttgt tattgttatt tgtggttttg 39000 gtgtcacatc taagaatctt ggcctaattc aaggtcatga agatttactc ttatgttttc 39060 ttctagatgt ttagttctat agttggagct catatattta ggtttctgat ccattttgag 39120 ttagtgtttg tataaagtgt gaggtagggg tccaacttca ttctttgaat gtgaatattc 39180 agttgttcca gcaccattag ttgagaagac cattctttcc ccattgaatg gtcctggcac 39240 ctattttatt aaatttaaat ctaaagaacc acatgtaggt tgggcacggt ggctcatgcc 39300 tgtaatccca acactttggg aggccaaggc tggtggatca cttgagctta ggagtttgag 39360 accagtttgg gcaacatagt ggaactctat ctctatcaaa aatacaaaaa atcagctggg 39420 catggtggta catgcctgta gtcccagcta ctagggaggc tgaggcagga aaatcgcctg 39480 agcccagcag gtagaggttg cagtgaactg agattgtgcc aatggactcc agcctgggtg 39540 acagaacaag acactacctc aaaaataaat aaatggataa ataaaaacca catgtgactg 39600 actactgtat tggatgtcac aaccctaggt tcaactgaag gaggtccaca cagcaccccc 39660 tgtgtataaa cagtcatgca catgcacgca cacacacaca cacacacaaa cacacacaca 39720 cagacacaaa gtgcttcccc attgcacaga gtcattttgc agatttgcac acacatggat 39780 ccagacacaa gtacttggat attcacggca ggcctgcctc ctctacccct aggccacatt 39840 ctagacaatt tctgcctccc tgacatgggg gccccaggac aggtgcctgg tcctgacctc 39900 tctccccttc atcctccagg gaaatcagcc ttacactggg ctgcggctgt gaacaacgtg 39960 gaagccactt tggccctgct caaaaatgga gccaataagg acatgcagga tagcaaggtg 40020 agccccagcc cttggtccac tgggtgtcag cagtggcaca gtgccattgc aatccagcct 40080 gggcaacaga gtgagactct ctcaaaaaac aaaacaaaat aaaaccccaa acattggatt 40140 aaaatataat ttactttggt gactaaagtt tttgggggcc ccttaaattt tgtgcctaat 40200 ggctgggtgt ggtggttcat gcctataatc ccagcacttt gggaggtcga gatgggtgga 40260 ttacttgagt tcaggagttt gagaccagcc tggccaacgt agtaaaaccc tgtctctatt 40320 aaaaatacaa aaattagctg ggcgtagtgg tgcacacctg tagtcccagc tgctcgggag 40380 gctgaggcag gagaatcgct tgaacccgga aggctgaggt tgcagtgaac tgaaatggcg 40440 ccactgcact ccagcctggg cgacacagtg agactctgtc aaaaaaaaaa aaaaaaaaag 40500 acaagaaaaa aaaagttatg cctaaggtga gtacctcgct taactcaccc tagtcctggc 40560 cttgacctct ggcacttagt aggtgatgga tgaatgtggt ttagaggaaa gaacttgtcc 40620 aggctccccc agcacagccg ggatttaacc caggtctgtc aagctccagt gtacaaactc 40680 atagctctcg ggctccccca agaggctgga agactttgct actgttagct ggggtttcgc 40740 tgacctctgt gggttctggc cccccaggag gagacccccc tattcctggc cgcccgcgag 40800 ggcagctatg aggctgccaa gctgctgttg gaccactttg ccaaccgtga gatcaccgac 40860 cacctggaca ggctgccgcg ggacgtagcc caggagagac tgcaccagga catcgtgcgc 40920 ttgctggatc aacccagtgg gccccgcagc ccccccggtc cccacggcct ggggcctctg 40980 ctctgtcctc caggggcctt cctccctggc ctcaaagcgg cacagtcggg gtccaagaag 41040 agcaggaggc cccccgggaa ggcggggctg gggccgcagg ggccccgggg gcggggcaag 41100 aagctgacgc tggcctgccc gggccccctg gctgacagct cggtcacgct gtcgcccgtg 41160 gactcgctgg actccccgcg gcctttcggt gggccccctg cttcccctgg tggcttcccc 41220 cttgaggggc cctatgcagc tgccactgcc actgcagtgt ctctggcaca gcttggtggc 41280 ccaggccggg cgggtctagg gcgccagccc cctggaggat gtgtactcag cctgggcctg 41340 ctgaaccctg tggctgtgcc cctcgattgg gcccggctgc ccccacctgc ccctccaggc 41400 ccctcgttcc tgctgccact ggcgccggga ccccagctgc tcaacccagg gacccccgtc 41460 tccccgcagg agcggccccc gccttacctg gcagtcccag gacatggcga ggagtacccg 41520 gcggctgggg cacacagcag ccccccaaag gcccgcttcc tgcgggttcc cagtgagcac 41580 ccttacctga ccccatcccc cgaatcccct gagcactggg ccagcccctc acctccctcc 41640 ctctcagact ggtccgaatc cacgcctagc ccagccactg ccactggggc catggccacc 41700 accactgggg cactgcctgc ccagccactt cccttgtctg ttcccagctc ccttgctcag 41760 gcccagaccc agctggggcc ccagccggaa gttaccccca agaggcaagt gttggcctga 41820 gacgctcgtc agttcttaga tcttgggggc ctaaagagac ccccgtcctg cctcctttct 41880 ttctctgtct cttccttcct tttagtcttt ttcatcctct tctctttcca ccaaccctcc 41940 tgcatccttg ccttgcagcg tgaccgagat aggtcatcag cccagggctt cagtcttcct 42000 ttatttataa tgggtggggg ctaccaccca ccctctcagt cttgtgaaga gtctgggacc 42060 tccttcttcc ccacttctct cttccctcat tcctttctct ctccttctgg cctctcattt 42120 ccttacactc tgacatgaat gaattattat tatttttatt tttctttttt tttttacatt 42180 ttgtatagaa acaaattcat ttaaacaaac ttattattat tattttttac aaaatatata 42240 tatggagatg ctccctcccc ctgtgaaccc cccagtgccc ccgtggggct gagtctgtgg 42300 gcccattcgg ccaagctgga ttctgtgtac ctagtacaca ggcatgactg ggatcccgtg 42360 taccgagtac acgacccagg tatgtaccaa gtaggcaccc ttgggcgcac ccactggggc 42420 caggggtcgg gggagtgttg ggagcctcct ccccacccca cctccctcac ttcactgcat 42480 tccagatggg acatgttcca tagccttgct ggggaagggc ccactgccaa ctccctctgc 42540 cccagcccca cccttggcca tctccctttg ggaactaggg ggctgctggt gggaaatggg 42600 agccagggca gatgtatgca ttcctttgtg tccctgtaaa tgtgggacta caagaagagg 42660 agctgcctga gtggtacttt ctcttcctgg taatcctctg gcccagcctc atggcagaat 42720 agaggtattt ttaggctatt tttgtaatat ggcttctggt caaaatccct gtgtagctga 42780 attcccaagc cctgcattgt acagcccccc actcccctca ccacctaata aaggaatagt 42840 taacactcag tgttgttggt ctgtgtctag gtaaggtggg gagtggtggc agtgggactt 42900 ctatctcccc cacccagggc taacttgagc tcccatcttg gggtaaatac atttgacttg 42960 ccagtctact tatgcttcct cttttggcag atgactaccg attggattag tggttgtcac 43020 ctgacttaag ctgagccaat cagattcttt tgctcgagaa ctttctttaa tggagaggct 43080 aagaaagttg tcagttggtg gagctcttaa ggtcacaatc agatttagaa atatcagtgg 43140 ccaattcgag gtggtgggca aagagacaag caaacagggc agaagaatga agctaatatt 43200 cagggagaat cagaaatgag agctcaaatg gctccttgag ggctgggggg gttatctcgg 43260 ctcccagtgc agttatcaat tccagttaat tgagtgttca ttccattgag atcaacaggt 43320 atttattaat tgctttctaa gtatctgatc atggttctgc atgaatttca cttttacttc 43380 atgctcctat gggttttgga gataaccttg gacccatgta ataaatactt ctttacttgt 43440 gccagcttcg gtgggtttct gttactcgca accagtcgtg ctccaagaca aggttctgtt 43500 tacactggtg tcttcaggaa aggaggatag gatttaatgt tcgttgattc tgtcagttgg 43560 agtgctcctg gttgcaaatg acacaaaagt ctactggctt aaacaagaaa ggggtttatt 43620 agctcatata aattaaaagg acctacttca ggggagaatt gatctagtgg ctcaaatgtg 43680 gtcaacaatg gctcagtttc tcttcatctc tacagctttc tctgggcatc agtttcatct 43740 acatgctcca catggcaccc agtagaacct ctcttcccga catcacacag ctccagcctt 43800 ctctctgtgt tatcaaaata ggtgccatat tgctccttcc agggaagaga gaaactccta 43860 gtattgtgat tgatttttct tttactttct tccttttttt tttttttttt ttaagataga 43920 gtcttgttct tgtcacccag gctggagtgc aatggcacaa tctcggctca ctgcatcccc 43980 cgcctccggg ttcaagcaat tctcctgcct cagtctccca agtagctggg attacaggtg 44040 tccacctcca tgcctggcta atttttgtat tttcagtaga gacaggattt caccatgttg 44100 gccaggctgg tctcgaactc cggacctcag gtgatccatc caccttggcc tcccaaagtg 44160 ctgggattac aggcatgagt catcgcactc gaccttgtgg ttgatttttc tattgactcc 44220 aattggttca cgtatccacc ctctaaccta aggtttctta atttcggcat tattgacatt 44280 tggggccaaa tcattctttg ctgtggggag ctgtcctgtg cattgtagga tatttaatag 44340 catctctg 44348 12 20 DNA Artificial Sequence Antisense Oligonucleotide 12 cccgctagca gcagcagcag 20 13 20 DNA Artificial Sequence Antisense Oligonucleotide 13 caacgacctc catttgcaca 20 14 20 DNA Artificial Sequence Antisense Oligonucleotide 14 gggtgcaacg acctccattt 20 15 20 DNA Artificial Sequence Antisense Oligonucleotide 15 gccaccactg aactctggca 20 16 20 DNA Artificial Sequence Antisense Oligonucleotide 16 ggacagtcgt ccacgttcac 20 17 20 DNA Artificial Sequence Antisense Oligonucleotide 17 aggagggcac tggcagttat 20 18 20 DNA Artificial Sequence Antisense Oligonucleotide 18 atattctgac tgcagctctc 20 19 20 DNA Artificial Sequence Antisense Oligonucleotide 19 cacaggaggc cagtcttgcc 20 20 20 DNA Artificial Sequence Antisense Oligonucleotide 20 tttgtgtcac agatagcatc 20 21 20 DNA Artificial Sequence Antisense Oligonucleotide 21 gtctcacagc gaggtccagt 20 22 20 DNA Artificial Sequence Antisense Oligonucleotide 22 gttcctgtga agcctgccat 20 23 20 DNA Artificial Sequence Antisense Oligonucleotide 23 tgcagctgaa gccattgact 20 24 20 DNA Artificial Sequence Antisense Oligonucleotide 24 ccacctggct ctcgcagcgt 20 25 20 DNA Artificial Sequence Antisense Oligonucleotide 25 gcagaggtac ttgtccacca 20 26 20 DNA Artificial Sequence Antisense Oligonucleotide 26 cagcggcaga ggtacttgtc 20 27 20 DNA Artificial Sequence Antisense Oligonucleotide 27 tcgcagttca cacctgtggt 20 28 20 DNA Artificial Sequence Antisense Oligonucleotide 28 cggttgatgc catcacggca 20 29 20 DNA Artificial Sequence Antisense Oligonucleotide 29 cactcattga tctccacgtt 20 30 20 DNA Artificial Sequence Antisense Oligonucleotide 30 ttttccccat ccacacagga 20 31 20 DNA Artificial Sequence Antisense Oligonucleotide 31 tccacatcct gctggcatcg 20 32 20 DNA Artificial Sequence Antisense Oligonucleotide 32 gtcgggcagg tcctgttcgc 20 33 20 DNA Artificial Sequence Antisense Oligonucleotide 33 ggcagtgggc tcctgtgtag 20 34 20 DNA Artificial Sequence Antisense Oligonucleotide 34 cgcctgacac agctgctcca 20 35 20 DNA Artificial Sequence Antisense Oligonucleotide 35 ctgggcacac gcagtagtgg 20 36 20 DNA Artificial Sequence Antisense Oligonucleotide 36 atatagccac ggcaggtccc 20 37 20 DNA Artificial Sequence Antisense Oligonucleotide 37 caggaagaca ctcacacatg 20 38 20 DNA Artificial Sequence Antisense Oligonucleotide 38 gaaaccaccc accaggtcca 20 39 20 DNA Artificial Sequence Antisense Oligonucleotide 39 cagtcccggg tgtgtgccgc 20 40 20 DNA Artificial Sequence Antisense Oligonucleotide 40 cggcactggc ctccatgctg 20 41 20 DNA Artificial Sequence Antisense Oligonucleotide 41 caggtgaagg tcagcccacc 20 42 20 DNA Artificial Sequence Antisense Oligonucleotide 42 agtgacaggt gaaggtcagc 20 43 20 DNA Artificial Sequence Antisense Oligonucleotide 43 tgcagctccc ggcaggagcg 20 44 20 DNA Artificial Sequence Antisense Oligonucleotide 44 ggcactgcag ctcccggcag 20 45 20 DNA Artificial Sequence Antisense Oligonucleotide 45 cagcctgggc tgttgcactc 20 46 20 DNA Artificial Sequence Antisense Oligonucleotide 46 gcagcggctg ttgttgaaga 20 47 20 DNA Artificial Sequence Antisense Oligonucleotide 47 gtggtcggcg cagtacttct 20 48 20 DNA Artificial Sequence Antisense Oligonucleotide 48 gcaaagtggt cggcgcagta 20 49 20 DNA Artificial Sequence Antisense Oligonucleotide 49 aaccagaggg tgctgtgctc 20 50 20 DNA Artificial Sequence Antisense Oligonucleotide 50 cccttggcca tgttcttcat 20 51 20 DNA Artificial Sequence Antisense Oligonucleotide 51 tctcaccctt ggccatgttc 20 52 20 DNA Artificial Sequence Antisense Oligonucleotide 52 tccagtctgt ggccacctcc 20 53 20 DNA Artificial Sequence Antisense Oligonucleotide 53 atgcctggct cctctacctt 20 54 20 DNA Artificial Sequence Antisense Oligonucleotide 54 agtgccatgg ctggtgccac 20 55 20 DNA Artificial Sequence Antisense Oligonucleotide 55 ctagctgatg tgtcatctgc 20 56 20 DNA Artificial Sequence Antisense Oligonucleotide 56 gtgtctgccc cagcatccag 20 57 20 DNA Artificial Sequence Antisense Oligonucleotide 57 gcgatgagct cttccaccat 20 58 20 DNA Artificial Sequence Antisense Oligonucleotide 58 ggctggcgat gagctcttcc 20 59 20 DNA Artificial Sequence Antisense Oligonucleotide 59 gcttggcagc ctcatagctg 20 60 20 DNA Artificial Sequence Antisense Oligonucleotide 60 cagcagcttg gcagcctcat 20 61 20 DNA Artificial Sequence Antisense Oligonucleotide 61 cactgggttg atccagcaag 20 62 20 DNA Artificial Sequence Antisense Oligonucleotide 62 cactgcagtg gcagtggcag 20 63 20 DNA Artificial Sequence Antisense Oligonucleotide 63 acccgcagga agcgggcctt 20 64 20 DNA Artificial Sequence Antisense Oligonucleotide 64 tgggaacccg caggaagcgg 20 65 20 DNA Artificial Sequence Antisense Oligonucleotide 65 cggaccagtc tgagagggag 20 66 20 DNA Artificial Sequence Antisense Oligonucleotide 66 gcgtctcagg ccaacacttg 20 67 20 DNA Artificial Sequence Antisense Oligonucleotide 67 caagatctaa gaactgacga 20 68 20 DNA Artificial Sequence Antisense Oligonucleotide 68 caaggcaagg atgcaggagg 20 69 20 DNA Artificial Sequence Antisense Oligonucleotide 69 ttcatgtcag agtgtaagga 20 70 20 DNA Artificial Sequence Antisense Oligonucleotide 70 catatatata ttttgtaaaa 20 71 20 DNA Artificial Sequence Antisense Oligonucleotide 71 cacagactca gccccacggg 20 72 20 DNA Artificial Sequence Antisense Oligonucleotide 72 atccagcttg gccgaatggg 20 73 20 DNA Artificial Sequence Antisense Oligonucleotide 73 tacctgggtc gtgtactcgg 20 74 20 DNA Artificial Sequence Antisense Oligonucleotide 74 gccccagtgg gtgcgcccaa 20 75 20 DNA Artificial Sequence Antisense Oligonucleotide 75 tggaatgcag tgaagtgagg 20 76 20 DNA Artificial Sequence Antisense Oligonucleotide 76 gtgggccctt ccccagcaag 20 77 20 DNA Artificial Sequence Antisense Oligonucleotide 77 agttcccaaa gggagatggc 20 78 20 DNA Artificial Sequence Antisense Oligonucleotide 78 tcccacattt acagggacat 20 79 20 DNA Artificial Sequence Antisense Oligonucleotide 79 tcaggcagct cctcttcttg 20 80 20 DNA Artificial Sequence Antisense Oligonucleotide 80 ccagaggatt accaggaaga 20 81 20 DNA Artificial Sequence Antisense Oligonucleotide 81 aaaaatacct ctattctgcc 20 82 20 DNA Artificial Sequence Antisense Oligonucleotide 82 ggctggtgga tcacctgagg 20 83 20 DNA Artificial Sequence Antisense Oligonucleotide 83 ctccgcctcc tgaggtcaag 20 84 20 DNA Artificial Sequence Antisense Oligonucleotide 84 gggatatgcc ttggattgag 20 85 20 DNA Artificial Sequence Antisense Oligonucleotide 85 atgggctcac ttgcaagtgc 20 86 20 DNA Artificial Sequence Antisense Oligonucleotide 86 ggtcactcac ccgatcacct 20 87 20 DNA Artificial Sequence Antisense Oligonucleotide 87 tgtcactaac ctgggccacg 20 88 20 DNA Artificial Sequence Antisense Oligonucleotide 88 ggatgagaat ctaggacaga 20 89 20 DNA Artificial Sequence Antisense Oligonucleotide 89 ggttttacta cgttggccag 20 90 20 DNA H. sapiens 90 ctgctgctgc tgctagcggg 20 91 20 DNA H. sapiens 91 tgtgcaaatg gaggtcgttg 20 92 20 DNA H. sapiens 92 tgccagagtt cagtggtggc 20 93 20 DNA H. sapiens 93 gtgaacgtgg acgactgtcc 20 94 20 DNA H. sapiens 94 ataactgcca gtgccctcct 20 95 20 DNA H. sapiens 95 gagagctgca gtcagaatat 20 96 20 DNA H. sapiens 96 ggcaagactg gcctcctgtg 20 97 20 DNA H. sapiens 97 gatgctatct gtgacacaaa 20 98 20 DNA H. sapiens 98 actggacctc gctgtgagac 20 99 20 DNA H. sapiens 99 atggcaggct tcacaggaac 20 100 20 DNA H. sapiens 100 agtcaatggc ttcagctgca 20 101 20 DNA H. sapiens 101 acgctgcgag agccaggtgg 20 102 20 DNA H. sapiens 102 tggtggacaa gtacctctgc 20 103 20 DNA H. sapiens 103 gacaagtacc tctgccgctg 20 104 20 DNA H. sapiens 104 accacaggtg tgaactgcga 20 105 20 DNA H. sapiens 105 tgccgtgatg gcatcaaccg 20 106 20 DNA H. sapiens 106 aacgtggaga tcaatgagtg 20 107 20 DNA H. sapiens 107 tcctgtgtgg atggggaaaa 20 108 20 DNA H. sapiens 108 cgatgccagc aggatgtgga 20 109 20 DNA H. sapiens 109 gcgaacagga cctgcccgac 20 110 20 DNA H. sapiens 110 ctacacagga gcccactgcc 20 111 20 DNA H. sapiens 111 tggagcagct gtgtcaggcg 20 112 20 DNA H. sapiens 112 ccactactgc gtgtgcccag 20 113 20 DNA H. sapiens 113 gggacctgcc gtggctatat 20 114 20 DNA H. sapiens 114 catgtgtgag tgtcttcctg 20 115 20 DNA H. sapiens 115 tggacctggt gggtggtttc 20 116 20 DNA H. sapiens 116 gcggcacaca cccgggactg 20 117 20 DNA H. sapiens 117 cagcatggag gccagtgccg 20 118 20 DNA H. sapiens 118 ggtgggctga ccttcacctg 20 119 20 DNA H. sapiens 119 cgctcctgcc gggagctgca 20 120 20 DNA H. sapiens 120 ctgccgggag ctgcagtgcc 20 121 20 DNA H. sapiens 121 gagtgcaaca gcccaggctg 20 122 20 DNA H. sapiens 122 tcttcaacaa cagccgctgc 20 123 20 DNA H. sapiens 123 agaagtactg cgccgaccac 20 124 20 DNA H. sapiens 124 tactgcgccg accactttgc 20 125 20 DNA H. sapiens 125 gagcacagca ccctctggtt 20 126 20 DNA H. sapiens 126 atgaagaaca tggccaaggg 20 127 20 DNA H. sapiens 127 gaacatggcc aagggtgaga 20 128 20 DNA H. sapiens 128 ggaggtggcc acagactgga 20 129 20 DNA H. sapiens 129 aaggtagagg agccaggcat 20 130 20 DNA H. sapiens 130 gtggcaccag ccatggcact 20 131 20 DNA H. sapiens 131 gcagatgaca catcagctag 20 132 20 DNA H. sapiens 132 ctggatgctg gggcagacac 20 133 20 DNA H. sapiens 133 atgaggctgc caagctgctg 20 134 20 DNA H. sapiens 134 ctgccactgc cactgcagtg 20 135 20 DNA H. sapiens 135 aaggcccgct tcctgcgggt 20 136 20 DNA H. sapiens 136 ccgcttcctg cgggttccca 20 137 20 DNA H. sapiens 137 ctccctctca gactggtccg 20 138 20 DNA H. sapiens 138 caagtgttgg cctgagacgc 20 139 20 DNA H. sapiens 139 tcgtcagttc ttagatcttg 20 140 20 DNA H. sapiens 140 cctcctgcat ccttgccttg 20 141 20 DNA H. sapiens 141 tccttacact ctgacatgaa 20 142 20 DNA H. sapiens 142 cccgtggggc tgagtctgtg 20 143 20 DNA H. sapiens 143 ccgagtacac gacccaggta 20 144 20 DNA H. sapiens 144 ttgggcgcac ccactggggc 20 145 20 DNA H. sapiens 145 cctcacttca ctgcattcca 20 146 20 DNA H. sapiens 146 cttgctgggg aagggcccac 20 147 20 DNA H. sapiens 147 gccatctccc tttgggaact 20 148 20 DNA H. sapiens 148 atgtccctgt aaatgtggga 20 149 20 DNA H. sapiens 149 caagaagagg agctgcctga 20 150 20 DNA H. sapiens 150 tcttcctggt aatcctctgg 20 151 20 DNA H. sapiens 151 ggcagaatag aggtattttt 20 152 20 DNA H. sapiens 152 cttgacctca ggaggcggag 20 153 20 DNA H. sapiens 153 gcacttgcaa gtgagcccat 20 154 20 DNA H. sapiens 154 cgtggcccag gttagtgaca 20 155 20 DNA H. sapiens 155 ctggccaacg tagtaaaacc 20 

What is claimed is:
 1. A compound 8 to 80 nucleobases in length targeted to a nucleic acid molecule encoding Notch3, wherein said compound specifically hybridizes with said nucleic acid molecule encoding Notch3 (SEQ ID: NO 4) and inhibits the expression of Notch3.
 2. The compound of claim 1 comprising 12 to 50 nucleobases in length.
 3. The compound of claim 2 comprising 15 to 30 nucleobases in length.
 4. The compound of claim 1 comprising an oligonucleotide.
 5. The compound of claim 4 comprising an antisense oligonucleotide.
 6. The compound of claim 4 comprising a DNA oligonucleotide.
 7. The compound of claim 4 comprising an RNA oligonucleotide.
 8. The compound of claim 4 comprising a chimeric oligonucleotide.
 9. The compound of claim 4 wherein at least a portion of said compound hybridizes with RNA to form an oligonucleotide-RNA duplex.
 10. The compound of claim 1 having at least 70% complementarity with a nucleic acid molecule encoding Notch3 (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of Notch3.
 11. The compound of claim 1 having at least 80% complementarity with a nucleic acid molecule encoding Notch3 (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of Notch3.
 12. The compound of claim 1 having at least 90% complementarity with a nucleic acid molecule encoding Notch3 (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of Notch3.
 13. The compound of claim 1 having at least 95% complementarity with a nucleic acid molecule encoding Notch3 (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of Notch3.
 14. The compound of claim 1 having at least one modified internucleoside linkage, sugar moiety, or nucleobase.
 15. The compound of claim 1 having at least one 2′-O-methoxyethyl sugar moiety.
 16. The compound of claim 1 having at least one phosphorothioate internucleoside linkage.
 17. The compound of claim 1 having at least one 5-methylcytosine.
 18. A method of inhibiting the expression of Notch3 in cells or tissues comprising contacting said cells or tissues with the compound of claim 1 so that expression of Notch3 is inhibited.
 19. A method of screening for a modulator of Notch3, the method comprising the steps of: a. contacting a preferred target segment of a nucleic acid molecule encoding Notch3 (SEQ ID NO: 4) with one or more candidate modulators of Notch3, and b. identifying one or more modulators of Notch3 expression which modulate the expression of Notch3.
 20. The method of claim 19 wherein the modulator of Notch3 expression comprises an oligonucleotide, an antisense oligonucleotide, a DNA oligonucleotide, an RNA oligonucleotide, an RNA oligonucleotide having at least a portion of said RNA oligonucleotide capable of hybridizing with RNA to form an oligonucleotide-RNA duplex, or a chimeric oligonucleotide.
 21. A diagnostic method for identifying a disease state comprising identifying the presence of Notch3 in a sample using at least one of the primers comprising SEQ ID NOs: 5 or 6, or the probe comprising SEQ ID NO:
 7. 22. A kit or assay device comprising the compound of claim
 1. 23. A method of treating an animal having a disease or condition associated with Notch3 comprising administering to said animal a therapeutically or prophylactically effective amount of the compound of claim 1 so that expression of Notch3 is inhibited.
 24. The method of claim 23 wherein the disease or condition is a hyperproliferative disorder. 